Mutations that confer genetic stability to genes in influenza viruses

ABSTRACT

The disclosure provides for an isolated recombinant influenza virus having at least one of: a PB2 viral segment encoding PB2 with residue at position 540 that is not asparagine or a residue at position 712 that is not glutamic acid, a PA viral segment encoding PA with a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or a PB1 viral segment encoding PB1 with a residue at position 149 that is not valine, a residue at position 634 that is not glutamic acid or a residue at position 635 that is not aspartic acid, or any combination thereof, and methods of making and using the virus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/749,910, filed on Jan. 22, 2020, which claims the benefit of thefiling date of U.S. application Ser. No. 62/795,821, filed on Jan. 23,2019, the disclosure of which is incorporated by reference herein.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under HHSN272201400008Cawarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

Influenza A virus is a respiratory pathogen that causes annual epidemicsand sporadic pandemics (Wright et al., 2013). Moreover, highlypathogenic avian H5N1 and the recently emerged H7N9 influenza viruseshave caused an appreciable number of human infections with highmortality rates (Watanabe et al., 2013; Zhang et al., 2013). Influenzaviruses infect respiratory epithelial cells and alveolar macrophages inmammalian hosts (Yu et al., 2010). The host immune system recognizes theRNA genome of influenza viruses via cytosolic sensors (Diebold et al.,2004; Pichlmair et al., 2006), which trigger innate immune responsesthat lead to the production of type I interferons (IFNs) andproinflammatory cytokines and chemokines (Honda and Taniguchi, 2006).Type I IFNs upregulate the production of antiviral proteins includingmyxovirus resistance (Mx), oligoadenylate synthetase (OAS), andinterferon-stimulated gene 15 (ISG15) (Garcia-Sastre et al., 2011).Dysregulation of the innate immune responses to influenza virusinfection causes lung pathology mediated by infiltrating immune cells,including macrophages and neutrophils (Heron et al., 2008; Perrone etal., 2008). Although several studies have addressed host responses toinfluenza virus infections (Fakuyama and Kawaoka, 2011), the mechanismsof influenza virus-induced pathology are still not fully understood.

To analyze the immune responses to influenza virus infection in vivo,viruses have been generated that expressed a fluorescent reporterprotein (Kittel et al., 2004; Shinya et al., 2004). However, theseviruses were significantly attenuated (Kittel et al., 2004; Shinya etal., 2004) and may not accurately reflect natural infections. Forexample, Manicassamy et al. (2010) generated a GFP-expressing influenzavirus, which they used to assess the route of antigen presentation uponinfluenza virus infection (Helft et al., 2012). However, the GFP genewas not stably maintained during replication in mouse lung or culturedcells (Manicassamy et al., 2010).

Highly pathogenic avian influenza viruses (HPAI) of the H5N1 subtypecontinue to evolve in nature, threatening animal and public health.These viruses were first identified in Guangdong province in China in1996 (Li et al., 2006), and have since been found in over 63 countriesin multiple avian species, repeatedly infecting mammals such as pigs andhumans (Li et al., 2010; Neumann et al., 2010). By December 2013, 648human cases of H5N1 virus infection had been confirmed by the Worldhealth organization (WHO), of which 384 were fatal, yielding a mortalityrate of almost 60% (http://www.who.int). In addition, novel subtypes ofinfluenza viruses, such as H7N9 and H10N8 virus, have spontaneouslyappeared and sporadically infected humans causing fatal outcomes (Chenet al., 2014; Li et al., 2013) (http://www.who.int). Thus, the currentthreat from influenza viruses reminds us of the urgent need to gain athorough understanding of their pathogenic mechanism in order to developmore effective strategies for control, including dynamic processes ofinfluenza virus infection and virus-target cells in vivo remain unclear.

SUMMARY

The present disclosure relates to mutations in influenza virus viralsegment(s) that increase genetic; stability, for instance, of anadditional, non-influenza viral gene, such as a “heterologous” genesequence that is inserted into one of the viral segments, e.g., fused toan intact or modified (for example, truncated or internally deleted)viral protein coding region, or that is present on an additional viralsegment. In one embodiment, one or more of the mutations may be employedto enhance the stability of influenza viruses that are not augmentedwith heterologous gene sequences. In one embodiment, the heterologousgene sequence is a marker gene, e.g., a fluorescent protein gene such asone for GFP, BFP, RFP, or YFP, a luciferase gene, a beta-glucuronidasegene, or beta-lactamase gene. In one embodiment, the heterologoussequence is for a prophylactic gene product. In one embodiment, theheterologous sequence encodes a therapeutic gene product.

As disclosed herein, influenza viruses expressing fluorescent proteinsof different colors (Color-flu viruses) were generated. Virusescontaining the foreign matter gene were passaged. Upon adaptation tomice, stable expression of the fluorescent proteins in infected animalsallowed their detection by different types of microscopy and by flowcytometry. The use of fluorescent influenza viruses, each of whichstably expresses one of four different fluorescent proteins, allows forsimultaneous monitoring and live imaging Using these viruses, severalstudies were performed to demonstrate the versatility of these viruses.For example, this system was used to analyze the progression of viralspread in mouse lungs, for live imaging of virus-infected cells, and fordifferential gene expression studies in virus antigen-positive and-negative live cells in the lungs of Color-flu-infected mice. Thus,Color-flu viruses are powerful tools to analyze virus infections at thecellular level in vivo to better understand influenza pathogenesis.Moreover, different stabilizing mutations in the resulting viruses wereidentified. These mutations include the T380A in HA protein (numberingis that for H1) and E712D in PB2 protein of A/PR/8/34 (H1N1) virus, andV25A, R443K, K737R and P167S amino acid replacements in the PB2, PA, PB1and NS1 proteins of A/Vietnam/1203/2004 (H5N1) virus, respectively. Theindividual mutations in the H5 virus alone resulted in the viruscontaining a foreign gene more stable in vitro, and the combination ofall of them provided even greater stability in vivo. These mutations areuseful for any HA/NA combination.

In one embodiment, a recombinant virus has one or more stabilizingmutations, e.g., one or more substitutions in one or more influenzavirus proteins that enhance the stability or replication (for instance,enhance the titer) of the recombinant virus with the one or moresubstitutions relative to a corresponding virus without the one or moresubstitutions (a parental virus) and/or one or more substitutions in oneor more influenza virus proteins that enhance the stability orreplication of a heterologous gene sequence present on one of the viralsegments in the recombinant virus relative to a corresponding viruswithout the one or more substitutions that has the heterologous genesequence in the respective viral segment and/or one or moresubstitutions in one or more influenza virus proteins that enhance thestability or replication of a heterologous gene sequence that is presenton an additional viral segment in the recombinant virus relative to acorresponding virus without the one or more substitution and that hasthe additional viral segment with the heterologous gene sequence. Theone or more substitutions include but are not limited to substitutionsin any of influenza PA, PA-X, PB1, PB1-F2, PB2, NP, NS1, NS2, M1, M2,NA, and/or HA (e.g., a HA of influenza A virus), substitutions encodedin the corresponding viral segments therefor (PA, PB1, PB2, NP, NS, M,NA, and/or HA), or a combination of substitutions in any one of thoseinfluenza virus proteins or genes, or a combination of one or moresubstitutions in two or more of those proteins or genes. In oneembodiment, the one or more substitutions that enhance the stability orreplication of an influenza virus are in the PA protein, e.g., asubstitution for glutamine at position 180, for threonine at position200, or for arginine at position 443 in PA (which is located on theprotein surface) that enhances, for example, RNA replication, PAproteolytic activity and/or interaction with one or more viral orcellular proteins. In one embodiment, the substitution for arginine atposition 443 in PA is a conservative substitution. In one embodiment,the substitution for arginine at position 443 in PA is anon-conservative substitution. In one embodiment, the substitution forglutamine at position 180 in PA is a conservative substitution. In oneembodiment, the substitution for glutamine at position 180 in PA is anon-conservative substitution. In one embodiment, the substitution forthreonine at position 200 in PA is a conservative substitution. In oneembodiment, the substitution for threonine at position 200 in PA is anon-conservative substitution. In one embodiment, the one or moresubstitutions that enhance the stability or replication of an influenzavirus are in the PB2 protein, e.g., a substitution for valine atposition 25, a substitution for asparagine at position 540, and/or forglutamic acid at position 712 in PB2 that, for example, enhancespolymerase activity, interaction with MAVS (for position 25) and/orprotein folding or stability (for position 712). In one embodiment, thesubstitution for valine at position 25 in PB2 is a conservativesubstitution. In one embodiment, the substitution for valine at position25 in PB2 is a non-conservative substitution. In one embodiment, thesubstitution for asparagine at position 540 in PB2 is a conservativesubstitution. In one embodiment, the substitution for asparagine atposition 540 in PB2 is a non-conservative substitution. In oneembodiment, the substitution for glutamic acid at position 712 in PB2 isa conservative substitution. In one embodiment, the substitution forglutamic acid at position 712 in PB2 is a non-conservative substitution.In one embodiment, the one or more substitutions that enhance thestability or replication of an influenza virus are in the PB1 protein,e.g., a substitution for valine at position 149, for lysine at position737, for glutamic acid at position 684, and/or for aspartic acid atposition 685, in PB1 (which may be located on the protein surface) that,for instance, alter polymerase or endonuclease activity. In oneembodiment, the substitution for lysine at position 737 in PB1 is aconservative substitution. In one embodiment, the substitution forlysine at position 737 in PB1 is a non-conservative substitution. In oneembodiment, the substitution for valine at position 149 in PB1 is aconservative substitution. In one embodiment, the substitution forvaline at position 149 in PB1 is a non-conservative substitution. In oneembodiment, the substitution for glutamic acid at position 684 in PB1 isa conservative substitution. In one embodiment, the substitution forglutamic acid at position 684 in PB1 is a non-conservative substitution.In one embodiment, the substitution for aspartic acid at position 685 inPB1 is a conservative substitution. In one embodiment, the substitutionfor aspartic acid at position 685 in PB1 is a non-conservativesubstitution. In one embodiment, the one or more substitutions thatenhance the stability or replication, e.g., by altering the interferoninterfering activity or transcription regulatory activity of NS1 of aninfluenza virus, are in the NS1 protein, e.g., a substitution forproline at position 167 in NS1 which may alter interaction with cellularproteins. In one embodiment, the substitution for proline at position167 in NS1 is a conservative substitution. In one embodiment, thesubstitution for proline at position 167 in NS1 is a non-conservativesubstitution. In one embodiment, the one or more substitutions thatenhance the stability or replication of an influenza virus are in the HAprotein, e.g., a substitution for threonine at position 380 in HA (whichis in an alpha helix of HA-2). In one embodiment, the substitution forthreonine at position 380 in HA is a conservative substitution. In oneembodiment, the substitution for threonine at position 380 in HA is anon-conservative substitution. In one embodiment, the residue atposition 443 in PA is K or H. In one embodiment, the residue at position737 in PB1 is H or R. In one embodiment, the residue at position 25 inPB2 is A, L, T, I, or G. In one embodiment, the residue at position 712in PB2 is D. In one embodiment, the residue at position 167 in NS1 is C,M, A, L, I, G or T.

The viruses may be employed as vaccines or as gene delivery vectors.

The vectors comprise influenza cDNA, e.g., influenza A (e.g., anyinfluenza A gene including any of the 18 HA or 11 NA subtypes), B or CDNA (see Fields Virology (Fields et al. (eds.), Lippincott, Williams andWickens (2006), which is specifically incorporated by reference herein).

In one embodiment, PB1, PB2, PA, NP, M, and NS encode proteins having atleast 80%, e.g., 90°/c, 92%, 95%, 97%, 98%, or 99%, including anyinteger between 80 and 99, contiguous amino acid sequence identity to, apolypeptide encoded by one of SEQ ID NOs:1-6 or 10-15, although thedisclosed positions and substitutions in viral proteins may be made in aviral segment from any influenza virus isolate or may be used to selectviral segments with specified residues at the one or more disclosedpositions. In one embodiment, PB1, PB2, PA, NP, M, and NS encodeproteins that are having at least 80%, e.g., 90%, 92%, 95%, 97%, 98%, or99%, including any integer between 80 and 99, contiguous amino acidsequence identity to, a polypeptide encoded by one of SEQ ID NOs:1-6 or10-15. In one embodiment, the influenza virus polypeptide has one ormore, for instance, 2, 5, 10, 15, 20 or more, conservative amino acidssubstitutions, e.g., conservative substitutions of up to 10% or 20% of2, 5, 10, 15, 20 or more, of a combination of conservative andnon-conservative amino acids substitutions, e.g., conservativesubstitutions of up to 10% or 20% of the residues, or relative to apolypeptide encoded by one of SEQ ID NOs:1-6 or 10-15, and has acharacteristic residue as described herein that provides for stability.

A recombinant influenza virus of the disclosure may be prepared byselecting viral segments for inclusion in a recombinant virus, such as areassortant virus, having one or more stabilizing mutations in one ormore influenza virus proteins. For example, a HA viral segment encodinga HA with a residue at position 380 that is not threonine may beselected; a PA viral segment encoding a PA with a residue at position443 that is not arginine may be selected; a PA viral segment encoding aPA with a residue at position 180 that is not glutamine may be selected;a PA viral segment encoding a PA with a residue at position 200 that isnot threonine may be selected; a PB1 viral segment encoding a PB1 with aresidue at position 737 that is not lysine may be selected; a PB1 viralsegment encoding a PB1 with a residue at position 149 that is not valinemay be selected; a PB1 viral segment encoding a PB1 with a residue atposition 684 that is not glutamic acid may be selected; a PB1 viralsegment encoding a PB1 with a residue at position 685 that is notaspartic acid may be selected; a PB2 viral segment encoding a PB2 with aresidue at position 25 that is not valine, a residue that is notasparagine at position 540, or a residue at position 712 that is notglutamic acid may be selected; a NS viral segment encoding a NS1 with aresidue at position 167 that is not proline may be selected; or anycombination thereof. In one embodiment, the residue at position 443 inPA is K or H. In one embodiment, the residue at position 180 in PA is R,K or H. In one embodiment, the residue at position 200 in PA is A, G, I,L or V. In one embodiment, the residue at position 737 in PB1 is H or R.In one embodiment, the residue at position 149 in PB1 is A, T, G, I orL. In one embodiment, the residue at position 684 in PB1 is D or N. Inone embodiment, the residue at position 685 in PB1 is E or Q. In oneembodiment, the residue at position 25 in PB2 is A, L, T, I, or G. Inone embodiment, the residue at position 712 in PB2 is D. In oneembodiment, the residue at position 540 in PB2 is K, R or H. In oneembodiment, the residue at position 167 in NS1 is S, C, M, A, L, I, G orT. In one embodiment, the residue at position 380 in HA is A, I, V, L orG.

In one embodiment, the influenza virus of the disclosure is arecombinant influenza virus having two or more of selected amino acidresidues at specified positions in one or more of PA, PB1, PB2, HA,and/or NS1. In one embodiment, the recombinant reassortant influenzavirus has a lysine or histidine at position 443 in PA, a histidine orarginine at position 737 in PB1, a leucine, isoleucine, threonine,alanine or glycine at position 25 in PB2 and/or an aspartic acid,histidine, arginine, lysine or asparagine at position 712 in PB2; aleucine, alanine, valine, isoleucine, or glycine at position 380 in HA,or serine, cysteine, methionine, alanine, valine, glycine, isoleucine orleucine at position 167 in NS1.

A recombinant influenza virus of the disclosure having an extra viralsegment with a heterologous gene sequence (a “9 segment” virus) whichvirus has enhanced stability and/or replication may be prepared byselecting viral segments for inclusion in the recombinant virus havingone or more of the stabilizing mutations in an influenza virus protein.For example, a HA viral segment encoding a HA with a residue at position380 that is not threonine may be selected; a PA viral segment encoding aPA with a residue at position 443 that is not arginine may be selected;a PB1 viral segment encoding a PB1 with a residue at position 737 thatis not lysine may be selected; a PB2 viral segment encoding a PB2 with aresidue at position 25 that is not valine or a residue at position 712that is not glutamic acid may be selected; a NS viral segment enclosinga NS1 with a residue at position 167 that is not proline may beselected; or any combination thereof. The extra viral segment may bederived from any of the naturally occurring viral segments. In oneembodiment, the residue at position 443 in PA is K or H. In oneembodiment, the residue at position 737 in PB1 is H or R. In oneembodiment, the residue at position 25 in PB2 is A, L, T, I, or G. Inone embodiment, the residue at position 180 in PA is R, K or H. In oneembodiment, the residue at position 200 in PA is A, G, I, L or V. In oneembodiment, the residue at position 149 in PB1 is A, T, G, I or L. Inone embodiment, the residue at position 684 in PB1 is D or N. In oneembodiment, the residue at position 685 in PB1 is E or Q. In oneembodiment, the residue at position 540 in PB2 is K, R or H. In oneembodiment, the residue at position 712 in PB2 is D. In one embodiment,the residue at position 167 in NS1 is C, M, A, L, I, G or T. Theheterologous gene sequence may be of length that results in the viralsegment with that heterologous gene sequence having a length that is upto 4 kb, 4.2 kb, 4.5 kb, 4.7 kb, 5 kb, 5.2 kb, 5.5 kb, 5.7 kb or 6 kb inlength. In one embodiment, the heterologous gene in the extra viralsegment replaces influenza virus protein coding sequences (e.g., thereis a deletion of influenza virus coding sequences without deletingencapsidation (incorporation) sequences in coding sequences that arelinked to encapsidation sequences in non-coding sequences at one or bothends of the viral segment). In one embodiment, the heterologous genesequence in the extra viral segment is in genomic orientation. In oneembodiment, the heterologous gene sequence in the extra viral segment isfused in frame to N-terminal influenza virus protein coding sequences.In one embodiment, the heterologous gene sequence in the extra viralsegment is fused in frame to C-terminal influenza virus protein codingsequences. The heterologous gene may encode a RNA, e.g., a microRNA, ora protein, e.g., a gene product that is prophylactic or therapeutic. Inone embodiment, the gene product is an antigen from a differentinfluenza virus isolate, or an antigen from a bacteria, a virus otherthan influenza virus, a parasite, or a fungus.

A recombinant influenza virus of the disclosure having a heterologousgene sequence in one of the eight viral segments (an “8 segment” virus)with enhanced stability and/or replication may be prepared by selectingviral segments for inclusion in the recombinant virus having one or moreof the stabilizing mutations in an influenza virus protein. For example,a HA viral segment encoding a HA with a residue at position 380 that isnot threonine may be selected; a PA viral segment encoding a PA with aresidue at position 443 that is not arginine may be selected; a PB1viral segment encoding a PB1 with a residue at position 737 that is notlysine may be selected; a PB2 viral segment encoding a PB2 with aresidue at position 25 that is not valine or a residue at position 712that is not glutamic acid may be selected; a NS viral segment enclosinga NS1 with a residue at position 167 that is not proline may beselected; or any combination thereof. In one embodiment, the residue atposition 443 in PA is K or H. In one embodiment, the residue at position737 in PB1 is H or R. In one embodiment, the residue at position 25 inPB2 is A, L, T, I, or G. In one embodiment, the residue at position 712in PB2 is D. In one embodiment, the residue at position 180 in PA is R,K or H. In one embodiment, the residue at position 200 in PA is A, G, I,L or V. In one embodiment, the residue at position 149 in PB1 is A, T,G, I or L. In one embodiment, the residue at position 684 in PB1 is D orN. In one embodiment, the residue at position 685 in PB1 is E or Q. Inone embodiment, the residue at position 540 in PB2 is K, R or H. In oneembodiment, the residue at position 167 in NS1 is C, M, A, L, I, G or T.

A recombinant influenza virus of the disclosure having a heterologousgene sequence in one of the influenza virus viral segments that alsolacks a viral segment (a “7 segment” virus), which virus has enhancedstability and/or replication, may be prepared by selecting viralsegments for inclusion in the recombinant virus having one or more ofthe stabilizing mutations in an influenza virus protein. For example, aHA viral segment encoding a HA with a residue at position 380 that isnot threonine may be selected; a PA viral segment encoding a PA with aresidue at position 443 that is not arginine may be selected; a PB1viral segment encoding a PB1 with a residue at position 737 that is notlysine may be selected; a PB2 viral segment encoding a PB2 with aresidue at position 25 that is not valine or a residue at position 712that is not glutamic acid may be selected; a NS viral segment enclosinga NS1 with a residue at position 167 that is not proline may beselected; or any combination thereof. The viral segment that is omittedmay be any one of the naturally occurring viral segments and optionallythe encoded protein is provided in trans. In one embodiment, the 7segment virus includes a PA viral segment, or the PA protein is providedin trans, and the residue at position 443 in PA is K or H. In oneembodiment, the 7 segment virus includes a PB1 viral segment, or the PB1protein is provided in trans, and the residue at position 737 in PB1 isH or R. In one embodiment, the 7 segment virus includes a PB2 viralsegment, or the PB2 protein is provided in trans, and the residue atposition 25 in PB2 is A, L, T, I, or G. In one embodiment, the 7 segmentvirus includes a PB2 viral segment, or the PB2 protein is provided intrans, and the residue at position 712 in PB2 is D. In one embodiment,the 7 segment virus includes a PA viral segment, or the PA protein isprovided in trans, and the residue at position 180 in PA is R, K or H.In one embodiment, the 7 segment virus includes a PA viral segment, orthe PA protein is provided in trans, and the residue at position 200 inPA is A, G, I, L or V. In one embodiment, the 7 segment virus includes aPB1 viral segment, or the PB1 protein is provided in trans, and theresidue at position 149 in PB1 is A, T, G, I or L. In one embodiment,the 7 segment virus includes a PB1 viral segment, or the PB1 protein isprovided in trans, and the residue at position 684 in PB1 is D or N. Inone embodiment, the 7 segment virus includes a PB1 viral segment, or theP protein is provided in trans, and the residue at position 685 in PB1is E or Q. In one embodiment, the 7 segment virus includes a PB2 viralsegment, or the PB2 protein is provided in trans, and the residue atposition 540 in PB2 is K, R or H. In one embodiment, the 7 segment virusincludes a NS viral segment, or the NS1 protein is provided in trans,and the residue at position 167 in NS1 is C, M, A, L, I, G or T. Theheterologous gene sequence may be of length that results in the viralsegment with that heterologous gene sequence having a length that is upto 4 kb, 4.2 kb, 4.5 kb, 4.7 kb, 5 kb, 5.2 kb, 5.5 kb, 5.7 kb or 6 kb inlength. In one embodiment, the heterologous gene replaces influenzavirus protein coding sequences (e.g., there is a deletion of influenzavirus coding sequences without deleting encapsidation (incorporation)sequences in coding sequences that are linked to encapsidation sequencesin non-coding sequences at one or both ends of the viral segment). Inone embodiment, the heterologous gene sequence in the extra viralsegment is in genomic orientation. In one embodiment, the heterologousgene sequence is fused in frame to N-terminal influenza virus proteincoding sequences. In one embodiment, the heterologous gene sequence inthe extra viral segment is fused in frame to C-terminal influenza virusprotein coding sequences. The heterologous gene may encode a RNA, e.g.,a microRNA, or a protein, e.g., a gene product that is prophylactic ortherapeutic. In one embodiment, the gene product is an antigen from adifferent influenza virus isolate, or an antigen from a bacteria, avirus other than influenza virus, a parasite, or a fungus.

The heterologous gene sequence may be inserted into any viral segment.The heterologous gene sequence may be of length that results in theviral segment with that heterologous gene sequence having a length thatis up to 4 kb, 4.2 kb, 4.5 kb, 4.7 kb, 5 kb, 5.2 kb, 5.5 kb, 5.7 kb or 6kb in length. In one embodiment, the heterologous gene replaces internalinfluenza virus sequences in the viral segment. In one embodiment, theinsertion of a heterologous gene sequence may result in a “knock-out” ofthe respective influenza virus gene product and to prepare such a virus,influenza virus protein(s) may be provided in trans to complement thattype of mutation. In one embodiment, the heterologous gene sequences arein addition to influenza virus coding sequences in the viral segment. Inone embodiment, the heterologous gene sequence is fused in frame toN-terminal influenza virus protein coding sequences. In one embodiment,the heterologous gene in is fused in frame to C-terminal influenza virusprotein coding sequences. The heterologous gene may encode a RNA or aprotein, e.g., a gene product that is prophylactic or therapeutic. Inone embodiment, the gene product is an antigen from a differentinfluenza virus isolate, an antigen from a bacteria, a virus other thaninfluenza virus, a parasite, or a fungus. In one embodiment, theheterologous gene sequence is in the NA viral segment. In oneembodiment, the heterologous gene sequence is in the HA viral segment.In one embodiment, the heterologous gene sequence is in the M viralsegment. In one embodiment, the heterologous gene sequence is in the NSviral segment. In one embodiment, the heterologous gene sequence is inthe NP viral segment, e.g., see Liu et al., 2012; Wang et al. 2010;Arilor et al., 2010; Dos Santos Afonso et al., 2005). In one embodiment,the heterologous gene sequence is in the PA viral segment. In oneembodiment, the heterologous gene sequence is in the PB1 viral segment.In one embodiment, the heterologous gene sequence is in the PB2 viralsegment. In one embodiment, the heterologous gene sequence is 5′ or 3′to, replaces at least some of or is inserted into, the PA codingsequence in the PA viral segment. In one embodiment, the heterologousgene sequence is 5′ or 3′ to, replaces at least some of or is insertedinto, the PB1 coding sequence in the PB1 viral segment. In oneembodiment, the heterologous gene sequence is 5′ or 3′ to, replaces atleast some of or is inserted into, the PB2 coding sequence in the PB2viral segment (see, e.g., Avilov et al. 2012). In one embodiment, theheterologous gene sequence is 5′ or 3′ to, replaces at least some of oris inserted into, the NS coding sequence in the NS viral segment(Manicassamy et al. 2010). In one embodiment, the heterologous genesequence is 5′ or 3′ to, replaces at least some of or is inserted into,the NS1 coding sequence in the NS viral segment. In one embodiment, theheterologous gene sequence is 5′ or 3′ to, replaces at least some of oris inserted into, the NS2 coding sequence in the NS viral segment. Inone embodiment, the heterologous gene sequence is 5′ or 3′ to, replacesat least some of or is inserted into, the HA coding sequence in the HAviral segment. In one embodiment, the heterologous gene sequence is 5′or 3′ to, replaces at least some of or is inserted into, the NA codingsequence in the NA viral segment (see, e.g., Perez et al. 2004). In oneembodiment, the heterologous gene sequence is 5′ or 3′ to, replaces atleast some of or is inserted into, the M1 coding sequence in the M viralsegment. In one embodiment, the heterologous gene sequence is 5′ or 3′to, replaces at least some of or is inserted into, the M2 codingsequence in the M viral segment (see, e.g., Wei et al. 2011).

Further provided is a vaccine comprising the recombinant virus of thedisclosure, e.g., a live attenuated vaccine or where the recombinantvirus is cold adapted, one or more vectors comprising one or more viralsegments with one or more of the disclosed substitutions, as well asmethods of making and using the recombinant virus. In one embodiment,the vector for vRNA production comprises a promoter such as a RNApolymerase I promoter, a RNA polymerase II promoter, a RNA polymeraseIII promoter, a T3 promoter or a T7 promoter.

Also provided is a method to generate influenza viruses with an alteredproperty, e.g., enhanced replication or stability, in a selected avianor mammalian host. The method includes serially passaging an isolate ofan influenza virus in an individual host organism, and identifyingindividual viruses with the altered property and optionally molecularlycharacterizing the individual viruses.

Further provided is a set of recombinant influenza viruses, each memberof the set encoding a distinct optically detectable marker, e.g., theopen reading frame of which is fused to the open reading frame of aninfluenza virus protein, the open reading frame of which is on a ninthviral segment for influenza A or B viruses, or the open reading frame ofwhich replaces at least a portion of one of the viral protein codingregions. For example, one of the members includes a luminescent proteingene, e.g., a luciferase gene, a fluorescent protein gene, for instance,green fluorescent protein gene, yellow fluorescent protein gene, or redfluorescent protein gene, photoprotein genes such as Aequorinphotoprotein gene or obelin photoprotein gene, chloramphenicalacetyltransferase gene, a phosphatase gene such as alkaline phosphatasegene, a peroxidase gene such as horseradish peroxidase gene,beta-galactosidase gene, beta-lactamase gene or beta-glucuronidase gene.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1G. Characteristics of mouse-adapted Venus−PR8 in mice. (A-F)Four B6 mice per group were intranasally inoculated with WT−PR8,WT−Venus−PR8, or MA−Venus−PR8. Body weight and survival of mice weremonitored for 14 days. (G) Lungs of animals infected with 10⁴ PFU of PR8or MA−Venus PR8 (three mice per group) were harvested at days 3, 5, and7 p.i. Virus titers were analyzed by use of a plaque assay in MDCKcells.

FIGS. 2A-2C. Distribution of Color-flu viruses in lungs. (A) Lungtissues were harvested from B6 mice at days 3 and 5 p.i. with Color-fluviruses (105 PFU of MA−eCFP, eGFP, Venus, and mCherry−PR8). The openreading frame (ORF) of the NS1 gene without a stop codon was fused withthe N-terminus of fluorescent reporter genes (Venus, eCFP, eGFP, andmCherry) via a sequence encoding the protein linker GSGG. Thefluorescent genes are followed by a sequence encoding the GSG linker, afoot-and-mouth virus protease 2A autoproteolytic site with 57nucleotides from porcine teschovirus-1, and by the ORF of NEP. Inaddition, silent mutations were introduced into the endogenous spliceacceptor site of the NS1 ORF to prevent splicing. Whole-mount images oftransparent lung tissues were obtained by using a fluorescentstereomicroscope. (B, C) B6 mice were intranasally inoculated with amixture of MA−eCFP, eGFP, Venus, and mCherry-PR8 (2.5×10⁴ PFU perstrain). Scale bar, 5 mm. (B) The sections of lungs at days 2 and 5 p.i.were analyzed by using an inverted fluorescence microscope with a NuanceFX multispectral imaging system with InForm software. Scale bar, 100 μm.(C) Enlarged images of the indicated area in (B) were unmixed andseparated into autofluorescence (AF), eCFP, eGFP, Venus, and mCherryfluorescence. Arrows in the merged image indicate cells infected withdifferent color variants of Color-flu viruses.

FIGS. 3A-3K. Analysis of macrophage infiltration. (A-D) Lung tissueswere harvested from PBS-inoculated mice (mock) or mice infected with 105PFU of MA−Venus−PR8 at day 2 p.i., and tissues were fixed and processedfor histological analysis. Sections were incubated with PE-anti-Mac3antibody to detect macrophages (red) and counterstained with Hoechst dye(blue) to visualize nuclei. The fluorescent signal of the Venus proteinis shown in green. The scale bar represents 200 μm. (E-H) Kinetics ofthe interaction between virus infected cells and lung macrophages.Images of eGFP positive cells (green) and CD11b+ macrophage (red) inlung tissue from naïve B6 mice (upper left panel) and B6 mice on day 3p.i. with 105 PFU MA−eGFP−PR8 (upper right panel) were obtained by usingtwo-photon microscope. Sequential images in lower panel (1-4) show anenlarged view of the box in the upper right panel. Arrowheads indicatethe blebbing of eGFP positive cells. Scale bar. 40 μm. (l) Infection ofmacrophages by influenza viruses. Single cell suspensions were obtainedfrom lungs of PBS-inoculated (mock) mice or mice infected with 105 PFUof MA−Venus−PR8 at day 3 p.i., stained with antibodies against CD45,CD11b, and F4/80, and analyzed by flow cytometry. The panel shows Venusexpression versus the CD11b staining profile from cells gated on F4/80and CD45 expression levels. (J) Gene expression analysis. Total RNA wasisolated from sorted macrophages of PBS-inoculated (naive) mice, andfrom sorted Venus-positive (Venus(+)) and Venus-negative (Venus(−))macrophages of mice inoculated with 105 PFU of MA−Venus−PR8 at day 3p.i. (9 mice per treatment), and microarray analysis was performed. (J)Differentially expressed (DE) transcripts were identified by comparinggene expression levels in naive macrophages with those in Venus(+)macrophages from infected mice. Likewise, gene expression levels werecompared for naive macrophages and Venus(−) macrophages obtained frominfected mice. DE transcripts were organized by hierarchical clusteringand each cluster was analyzed for enriched biological functions. A heatmap of the clustered transcripts for each condition is displayed (acolor key is shown at the top of the panel), and the different clustersare illustrated by the color bar on the left of the heat map. Enrichedannotations for each cluster are listed to the left of each cluster,with the enrichment score for each annotation in parentheses. The blueline in the heat map illustrates fold changes of DE transcripts whencomparing Venus(+) with Venus(−) macrophages. A shift of the blue lineto the left indicates that the DE transcript is more highly expressed inVenus(+) macrophages, whereas a shift to the right indicates that the DEtranscript is more highly expressed in Venus(−) macrophages. (K) Thispanel shows a heat map comparing expression levels of type I interferons(IFNs) between Venus(+) and Venus(−) macrophages. A color key is shownat the bottom of the panel. NS denotes comparisons that were notstatistically significant between Venus(−) cells from infected animalsand naive macrophages from uninfected animals.

FIGS. 4A-4E. Characterization of MA−Venus−HPAI virus. (A) Four B6 miceper group were intranasally inoculated with MA−Venus−HPAI virus. Mousebody weight and survival were monitored for 14 days. (B) Lungs, spleens,kidneys, and brains were harvested from B6 mice at day 3 p.i. with 105PFU of MA−Venus−HPAI virus. Virus titers of tissue homogenates weredetermined by use of plaque assays in MDCK cells. Each data pointrepresents mean±s.d. (n=3) (C, D) Lung tissues were harvested from B6mice at day 1 and day 2 p.i. with 105 PFU of MA−Venus−HPAI virus andPR8. Images of transparent lung tissues (bronchus, red; alveolar, green)were obtained by a two-photon microscope. Each data point representsmean±s.d. (n=3). Statistical significance was calculated using theStudent's t-test. (D) The distribution of Venus-positive cells wasevaluated via volume analysis of the Venus-positive bronchus andalveolar area using 3D images of the transparent lung tissues. (E) Cellswere collected from lungs of B6 mice at days 1, 2, 3, and 4 p.i. with105 PFU of MA−Venus−PR8 or MA−Venus−HPAI virus, and stained for CD45,CD11b, and F4/80. Venus expression in CD45-negative cells, and the Venusversus F4/80 staining profile gated on CD45-positive cells were analyzedby flow cytometry. A representative data plot form day 2 p.i. is shownwith the percentage of Venus-positive cells.

FIG. 5. Virus yield of various viruses.

FIGS. 6A-6D. Virulence of WT−Venus−H5N1 virus and RG−MA virus in mice.Groups of four mice were intranasally infected with WT−Venus−H5N1 virusat doses of 101 to 105 PFU or with RG−MA virus at doses of 10⁰ to 10⁵PFU, and their body weight changes (A-C) and survival (B-D) weremonitored for two weeks.

FIG. 7. Venus expression of various H5N1 viruses in MDCK cells. MDCKcells were infected with Venus−H5N1-related viruses, and at 24 hpi theVenus expression of each virus plaque was observed by using fluorescentmicroscopy (Axio Observer.Z1, Zeiss). A representative image of eachvirus is shown.

FIG. 8. Venus expression of various H5N1 viruses in mouse lung. Groupsof three mice were intranasally infected with 105 PFU (50 μl) of virus.The mice were euthanized on day 2 p.i., and their lungs were collectedand fixed in 4% PFA and then embedded in O.C.T Compound. The frozentissues were cut into 5-μm slices and then stained with Hoechst 33342.Venus signal was detected by using the Nikon confocal microscope systemA1⁺. Blue represents nuclei stained by Hoechst 33342; green representsVenus expression.

FIG. 9. Genotypes of Venus−H5N1-related reassortants and their virulencein mice. The colors indicate the origins of the viral segments: blue,WT−Venus−H5N1 virus; red, MA−Venus−H5N1 virus. MLD₅₀ values weredetermined by inoculating groups of four mice with 10-fold serialdilutions contain 10⁰ to 105 PFU of virus in a 50-μL volume and werecalculated by using the method of Reed and Muench (30).

FIG. 10. Growth kinetics of reassortants in MDCK cells. MDCK cells wereinfected with virus at an MOI of 0.0001, and culture supernatants werecollected at the indicated times and then titrated in MDCK cells. Thereported values are means±standard deviations (SD) from two independentexperiments. *, P<0.01 compared with that of WT−Venus−H5N1virus-infected cells.

FIG. 11. Polymerase activity of different RNP combinations derived fromthe WT−Venus−H5N1 and MA−Venus−H5N1 viruses. 293 cells were transfectedin triplicate with a luciferase reporter plasmid and an internal controlplasmid, together with plasmids expressing PB1, PB2, PA, and NP fromeither WT−Venus−H5N1 or MA−Venus−H5N1 virus. Segments derived fromWT−Venus−H5N1 virus are shown in white, whereas those derived fromMA−Venus−H5N1 virus are in green. Cells were incubated at 37° C. for 24hours, and cell lysates were analyzed to measure firefly and Renillaluciferase activities. The values shown are means±SD of the threeindependent experiments and are standardized to the activity ofWT−Venus−H5N1 (100%). *, P<0.05 compared with that of WT−Venus−H5N1virus. *, P<0.01 compared with that of WT−Venus−H5N1 virus.

FIG. 12A. Venus-NS and deleted NS segments of Venus−H5N1-relatedreassortants. Viruses were passaged five times in MDCK cells and thevRNAs from the fifth passages were extracted by using a QIAamp® ViralRNA Mini Kit (QIAGEN). The respective NS segments were then amplified byusing PCR with NS-specific primers and run on an agarose gel. Lane 1,WT+MA−NS; lane 2, WT+MA−M; lane 3, WT+MA−NA; lane 4, WT+MA−PA; lane 5,WT+MA−PB1; lane 6, WT+MA−PB2; lane 7, WT+MA−(PB2+PA); lane 8,WT−Venus−H5N1; lane 9, RG−MA; lane 10, PR8; and lane 11, 1-kb DNAmarker.

FIG. 12B. Schematic of deleted viruses.

FIG. 13. High expression of Venus reassortants in mouse lung.

FIG. 14. Comparison of the growth capabilities of mutant viruses in MDCKcells. MDCK cells were infected at a MOI of 0.001 with PR8, NS1−VenusPR8 WT, NS1−Venus PR8 MA, and mutant NS1−Venus PR8 viruses that possessamino acid substitutions found in NS1−Venus PR8 MA virus. Virus titerswere determined every 12 hours by means of plaque assays. Results areexpressed as the mean titer (log₁₀ [PFU/ml])±standard deviation.

FIG. 15. Body weight changes and survival rates for mice infected withviruses carrying Venus. Four mice per group were intranasally infectedwith 10³, 10⁴ and 10⁵ PFU of each NS1−Venus PR8 virus. Body weights weremeasured and survival rates were monitored for 14 days after infection.

FIGS. 16A-16B. Virus titers in mouse lung. Nine mice per group wereintranasally infected with 103 PFU of PR8 (A) or the respectiveNS1−Venus PR8 virus (B). Three mice per group were euthanized on days 3,5, and 7 after infection and their lungs collected to determine virustiters. Virus titers were determined by means of plaque assays. Resultsare expressed as the mean of the titer (log₁₀ PFU/g)±standard deviation.Statistical significance was calculated by using the Tukey-Kramermethod. Asterisks indicate significant differences from titers from miceinfected with PR8 or NS1−Venus WT virus (P<0.05). ND: Not detected(detection limit, 5 PFU/lung).

FIG. 17. The stability of Venus expression by NS1−Venus PR8 MA virus invitro and in vivo. The positive rate of Venus expression was examined inMDCK cells and in mouse lung. Left panel: MDCK cells were infected withNS1−Venus PR8 MA virus at an MOI of 0.001, and supernatants werecollected every 24 hours. The positive rate of Venus expression wasestimated by dividing the number of plaques that expressed Venus by thetotal number of plaques. Middle panel: NS1−Venus PR8 MA virus wasserially passaged in MDCK cells five times and the positive rates ofVenus expression were estimated. Right panel: Nine mice were infectedwith 103 PFU of NS1−Venus PR8 MA virus. Three mice were euthanized ateach time point and plaque assays were performed using lung homogenates.The positive rates of Venus expression were estimated as describedabove.

FIGS. 18A-18G. Comparison of Venus expression in cells infected witheach NS1−Venus PR8 virus. (A-B) Venus protein expression in cellsinfected with each NS1−Venus PR8 virus was detected by means of westernblotting. MDCK cells were infected with each virus at an MOI of 1.Twelve hours after infection, virus-infected cells were lysed andwestern blotting was performed. An anti-GFP antibody was used to detectVenus protein, and M1 protein was detected as a control. The bandsappeared at approximately 27 kDa were shown in M1 panel. Representativeresults of two independent experiments are shown. (C-F) Observation ofVenus expression by use of confocal microscopy. MDCK cells were infectedwith each virus at an MOI of 1. Twelve hours after infection, cells werefixed, and Venus expression was observed. Representative results of twoindependent experiments are shown. Indicated viruses were used to infectMDCK cells (MOI of 1) and confocal microscopy was performed 12 hourslater. (G) HEK293 cells were infected with viral protein expressionplasmids for NP, PA, PB1 and PB2 or PB-2−E712D, together with a plasmidexpressing a vRNA encoding firefly luciferase.

FIG. 19. Polykaryon formation by HEK293 cells infected with wild-typePR8 or PR8 that possesses the HA−T380A mutation after exposure to low pHbuffer. The threshold for membrane fusion was examined at a pH range of5.5-5.9. HEK293 cells were infected with PR8 or PR8 that possesses theHA−T380A substitution. Eighteen hours after infection, HA on the cellsurface was digested with TPCK-trypsin, and exposure to the indicated pHbuffer. After fixation with methanol, the cells were stained withGiemsa's solution. Representative pictures are shown.

FIG. 20. Time-course observation of Venus-expressing cells intransparent lungs. Venus-expressing cells in whole lung lobe wereobserved. Three mice per group were intranasally infected with NS1-VenusPR8 MA, NS1−Venus PR8 WT or PR8 virus and lungs were collected on theindicated days. Mock-treated lungs served as a negative control. Toimage Venus-expressing cells deeper, lung samples were treated withSCALEVIEW A2, which make samples transparent, and were separated intoeach lobe and observed by using a stereo fluorescence microscope. Afterimaging the whole lung lobe (intact), samples were dissected to exposurethe bronchi (cut). Samples from mice infected with PR8 or NS1−Venus PR8WT virus were prepared on day 3 post-infection to compare with NS1−VenusPR8 MA virus-infected lungs in which the Venus signal was the brightestduring infection. Representative images are shown.

FIGS. 21A-21B. Analysis of Venus expression in CC10⁺ cells and SP-C⁺cells in lungs. Lung sections from mice infected with NS1−Venus PR8 MAvirus were stained with several antibodies specific for the epithelialcells in the lung. Mice were infected with 10⁴ PFU of NS1−Venus PR8 MAvirus and lungs were collected at 3 and 5 days post-infection. (A) Lungsection of mice infected with NS1−Venus PR8 MA virus were prepared at 3days post-infection and stained with an anti-CC10 polyclonal antibody(red). Scale bar: 100 μm. (B) Lung section of mice infected withNS1−Venus PR8 MA virus were prepared at 5 days post-infection andstained with an anti-SP-C polyclonal antibody (cyan) and ananti-podoplanin (Pdpn) polyclonal antibody (red). Venus-positive cellsin the alveolar region comprised SP-C-positive cells (white arrowhead)and podoplanin-positive cells (white arrow). Scale bar: 50 μm.

FIGS. 22A-22E. Flow cytometric analysis of Venus-positive cells inspecific cell types of the lung. Venus-positive cells in the indicatedcell types were analyzed by using flow cytometry. Mice were infectedwith 105 PFU of PR8 or NS1−Venus PR8 MA virus and lungs were collectedat 3 and 5 days post-infection. Single cell suspensions were stainedwith antibodies. (A) Representative dot plot for CD45⁺ live cells fromthe lung of mice inoculated with PBS are shown. (B, C) Total numbers ofeach specific cell species at the indicated time points are shown.Results are expressed as the mean cell numbers per lung±standarddeviation. CD45⁺ and via-probe⁻ cells were analyzed for monocytes andalveolar macrophages. (D, E) The numbers of Venus-positive cells incells defined in A and B at the indicated time points are shown. Resultsare expressed as the mean cell numbers±standard deviation. AM: alveolarmacrophage.

FIGS. 23A-23B. Sorting strategy to collect Venus-positive andVenus-negative cells in the F4/80⁺ population. Mice were infected with10⁵ PFU of NS1−Venus PR8 MA virus and lungs were collected at 3 dayspost-infection. Single cell suspensions were stained with a set ofantibodies. Lungs from mice inoculated with PBS were similarly stainedto confirm the autofluorescence of alveolar macrophages. (A)Representative dot plots showing the gating strategy to collectVenus-positive and -negative cells in a population of CD45⁺, via-probe⁻F4/80⁺ cells. The Venus-positive gate was shown not to include alveolarmacrophages. (B) Venus-positive and -negative cells collected from thelungs of mice infected with NS1−Venus PR8 MA virus were observed byusing an immunofluorescence assay.

FIGS. 24A-24D. Genes differentially expressed between Venus-positive and-negative F4/80⁺ cells. Mice were infected with 10⁵ PFU of NS1−Venus PR8MA virus and lungs were collected at 3 days post-infection. Single cellsuspensions were stained in the same manner as described in FIG. 10.Venus-positive and -negative cells were separately harvested by usingFACSAria II and subjected to microarray analysis. F4/80⁺ cells isolatedfrom the lungs of mice inoculated with PBS were used as a control. (A) Atotal of 633 genes were selected by student's T test (P<0.05) and byfiltering the genes whose expression changed at least 4.0-fold betweenthe Venus-positive and -negative groups from the genes whose expressionchanged at least 2.0-fold from the level of the PBS group. (B) Theseselected genes were functionally annotated by using Gene Ontology (GO)grouping. Statistical significance were determined by using Fisher'sexact test (P<0.01). (C) Hierarchical analysis of genes annotated in“cytokine activity” enriched by genes that were significantlydifferentially expressed between Venus-positive and -negative F4/80⁺cells. (D) Hierarchical analysis of genes annotated in “response towounding” enriched by genes that were significantly differentiallyexpressed between Venus-positive and -negative F4/80⁺ cells.

FIGS. 25A-25M. Exemplary parental sequences for PR8HG and the Cambridgestrain of PR8 (SEQ ID Nos: 1-19).

FIG. 26. Schematic of fusion protein comprising a heterologous protein.

FIG. 27. Schematic of mutations in polymerase complex proteins thatstabilize heterologous gene products mapped on the structure of thecomplex (PDB ID:4WSB).

FIGS. 28A-28C. A) Schematic structure of the eight viral RNA segmentscontained in WT−Venus−PR8. 2A, protease 2A autoproteolytic site. (B)Each virus was passaged in MDCK cells. The proportion ofVenus-expressing plaques in virus stocks from different passages wasdetermined in MDCK cells by using fluorescence microscopy. (C) The virusstocks from different passages were titrated by use of plaque assays inMDCK cells.

FIGS. 29A-29B. Effect of PB2−E712D on the mutation rate. (A) Each viruswas passaged five times in MDCK cells, and the mutations introduced intoeach segment during the passages were counted. (B) The mutation numberper nucleotide in each segment was calculated, and the mean values forall eight segments are shown.

FIGS. 30A-30F. RNA and protein expression in infected cells. (A to C)MDCK cells were infected with each virus at an MOI of 1. The relativeexpression levels of IFN-β mRNA (A), NS vRNA (B), and NP vRNA (C) weredetermined by quantitative real-time PCR at 9 h postinfection. (D) TheNS vRNA/NP vRNA ratio was calculated. (E) MDCK cells were infected withWT−Venus−PR8 (WT) or Venus−PR8−PB2−E712D (712) at an MOI of 1. Cellswere lysed at the indicated time points, and the expression of NS1, NP,and β-actin was detected by Western blotting. (F) NS1/NP ratios weredetermined based on the band intensity of the Western blotting.Means±the standard deviations of triplicate experiments, taking eachvalue in Venus−PR8-PB2−E712D-infected cells as 1, are shown in panels A,B. C, D, and F. *, P<0.01; ns, not significant (Student t test); hpi,hours postinfection.

FIGS. 31A-31C. Internal deletions occurred in the NS segment ofWT−Venus−PR8. (A) Schematic sequence of the NS segment in WT−Venus−PR8viruses that lost Venus expression after serial passages in MDCK cells.Selected examples are shown. (B) The procedure for the coinfectionexperiment is illustrated. The synonymous mutation was introduced intothe 3′ or 5′ region of the NS segment of WT−Venus−PR8. The viruses werethen used to coinfect MDCK cells. Viruses not expressing Venus wereplaque purified, and the sequences of their NS segments were analyzed.(C) Examples of the sequences of the NS segment of Venus-negativeviruses obtained after coinfection experiments. The red “X” indicates anintroduced synonymous mutation.

FIGS. 32A-32D. Additional mutations that stabilizes the Venus geneinserted into the NS segment. (A) Identified amino acid mutations weremapped onto the influenza polymerase complex (PDB ID 4WSB). (B)Polymerase internal tunnels (shown as yellow tubes). The vRNA promoterbinds to the polymerase, and the template vRNA enters the polymerasecomplex. The template vRNA passes through the active site, where RNAsynthesis occurs, and then leaves via the template exit. The RNAproducts synthesized at the active site, leave via the product exit. (C)Each mutant Venus−PR8 virus was passaged four times in MDCK cells, andthe proportion of Venus-expressing plaques after passaging wasdetermined in MDCK cells by using fluorescence microscopy. (D)Percentages of influenza A virus strains containing mutations thatstabilize the Venus gene in Venus−PR8 (i.e., the number of strainscontaining the indicated amino acid/total number of strains available inthe Influenza Research Database).

DETAILED DESCRIPTION Definitions

As used herein, the term “isolated” refers to in vitro preparationand/or isolation of a nucleic acid molecule, e.g., vector or plasmid,peptide or polypeptide (protein), or virus of the disclosure, so that itis not associated with in vivo substances, or is substantially purifiedfrom in vitro substances. An isolated virus preparation is generallyobtained by in vitro culture and propagation, and/or via passage ineggs, and is substantially free from other infectious agents.

As used herein, “substantially purified” means the object species is thepredominant species, e.g., on a molar basis it is more abundant than anyother individual species in a composition, e.g., is at least about 80%of the species present, and optionally 90% or greater, e.g., 95%, 98%,99% or more, of the species present in the composition.

As used herein, “substantially free” means below the level of detectionfor a particular infectious agent using standard detection methods forthat agent.

A “recombinant” virus is one which has been manipulated in vitro, e.g.,using recombinant DNA techniques, to introduce changes to the viralgenome. Reassortant viruses can be prepared by recombinant ornonrecombinant techniques.

As used herein, the term “recombinant nucleic acid” or “recombinant DNAsequence or segment” refers to a nucleic acid, e.g., to DNA, that hasbeen derived or isolated from a source, that may be subsequentlychemically altered in vitro, so that its sequence is not naturallyoccurring, or corresponds to naturally occurring sequences that are notpositioned as they would be positioned in the native genome. An exampleof DNA “derived” from a source, would be a DNA sequence that isidentified as a useful fragment, and which is then chemicallysynthesized in essentially pure form. An example of such DNA “isolated”from a source would be a useful DNA sequence that is excised or removedfrom said source by chemical means, e.g., by the use of restrictionendonucleases, so that it can be further manipulated, e.g., amplified,for use in the invention, by the methodology of genetic engineering.

As used herein, a “heterologous” influenza virus gene or viral segmentis from an influenza virus source that is different than a majority ofthe other influenza viral genes or viral segments in a recombinant,e.g., reassortant, influenza virus.

The terms “isolated polypeptide”, “isolated peptide” or “isolatedprotein” include a polypeptide, peptide or protein encoded by cDNA orrecombinant RNA including one of synthetic origin, or some combinationthereof.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule expressed from a recombinant DNAmolecule. In contrast, the term “native protein” is used herein toindicate a protein isolated from a naturally occurring (i.e., anonrecombinant) source. Molecular biological techniques may be used toproduce a recombinant form of a protein with identical properties ascompared to the native form of the protein.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Alignments using these programs can be performed using the defaultparameters. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). The algorithm may involve firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold. These initial neighborhood word hits act as seedsfor initiating searches to find longer HSPs containing them. The wordhits are then extended in both directions along each sequence for as faras the cumulative alignment score can be increased. Cumulative scoresare calculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when the cumulative alignmentscore falls off by the quantity X from its maximum achieved value, thecumulative score goes to zero or below due to the accumulation of one ormore negative-scoring residue alignments, or the end of either sequenceis reached.

In addition to calculating percent sequence identity, the BLASTalgorithm may also perform a statistical analysis of the similaritybetween two sequences. One measure of similarity provided by the BLASTalgorithm may be the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a test nucleicacid sequence is considered similar to a reference sequence if thesmallest sum probability in a comparison of the test nucleic acidsequence to the reference nucleic acid sequence is less than about 0.1,less than about 0.01, or less than about 0.001.

The BLASTN program (for nucleotide sequences) may use as defaults awordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5,N=−4, and a comparison of both strands. For amino acid sequences, theBLASTP program may use as defaults a wordlength (W) of 3, an expectation(E) of 10, and the BLOSUM62 scoring matrix. Seehttp://www.ncbi.n1m.nih.gov. Alignment may also be performed manually byinspection.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

“Conservative” amino acid substitutions refer to the interchangeabilityof residues having similar side chains. For example, a group of aminoacids having aliphatic side chains is glycine, alanine, valine, leucine,and isoleucine; a group of amino acids having aliphatic-hydroxyl sidechains is serine and threonine; a group of amino acids havingamide-containing side chains is asparagine and glutamine; a group ofamino acids having aromatic side chains is phenylalanine, tyrosine andtryptophan; a group of amino acids having basic side chains is lysine,arginine and histidine; and a group of amino acids havingsulfur-containing side chain is cysteine and methionine. In oneembodiment, conservative amino acid substitution groups are:threonine-valine-leucine-isoleucine-alanine; phenylalanine-tyrosine;lysine-arginine; alanine-valine; glutamic-aspartic; andasparagine-glutamine.

Encapsidation Sequences

The viral segment for incorporation of heterologous gene sequences intoa recombinant influenza virus includes at each end non-coding sequencesthat provide for encapsidation (incorporation or packaging) intovirions. The viral segments also include adjacent coding sequences, fromone or both ends, that contribute to encapsidation, e.g., enhanceencapsidation relative to viral segments that lack the adjacent codingsequences but have with the heterologous gene sequence. The vectors withthe viral segment having the heterologous gene sequence thus includeencapsidation sequences that at the 3′ end of vRNA that may includeadjacent 5′ coding sequences, at the 5′ end of vRNA that may include 3′coding sequences, or at the 3′ end of vRNA that may include adjacent 5′coding sequences and at the 5′ end of vRNA that may include 3′ codingsequences. For example, HA encapsidation sequences include sequences atthe 3′ end of HA vRNA including 33-nt of non-coding sequences and atleast 3, 6, 9, or 15 or up to about 216 nt of HA coding sequence and/orat the 5′ end of HA vRNA including about 45 nt of non-coding sequenceand up to about 75, 80, 268 or 291 of HA coding sequence (Watanabe etal. 2003). HS encapsidation sequences include sequences at the 3′ end ofNS vRNA including at least 30, 60, 90 or 150 nt of coding sequence andat the 5′ end of NS vRNA including at least 30, 60, 90 or 100 nt ofcoding sequence (Fujii et al. 2005).

In one embodiment, the 3′ NA incorporation sequences correspond tonucleotides 1 to 183, nucleotides 1 to 90, nucleotides 1 to 45,nucleotides 1 to 21, nucleotides 1 to 19 or any integer between 19 and183, of the N-terminal NA coding region, and may include a mutation atthe NA initiation codon. In another embodiment, the 5′ NA incorporationsequences correspond to sequences in the C-terminal coding region of NA,sequences corresponding to the 3′ most 39, 78, or 157, or any integerbetween 1 and 157, nucleotides for C-terminal NA coding region.

In one embodiment, the 5′ HA incorporation sequences correspond tosequences in the C-terminal coding region of HA, sequences correspondingto the 3′ most 75, 80, 268, 291, or 518, or any integer between 1 and518, nucleotides of the C-terminal HA coding region. The 3′ HAincorporation sequences correspond to nucleotides 1 to 3, 1 to 6, 1 to9, 1 to 15, 1 to 216, 1 to 468, or any integer between 1 and 468, of theN-terminal HA coding region.

In one embodiment, the 3′ PB1 or PB2 incorporation sequences correspondto nucleotides 1 to 250, nucleotides 1 to 200, nucleotides 1 to 150,nucleotides 1 to 160 or 1 to 130 or any integer between 1 and 250, ofthe N-terminal PB1 or PB2 coding region. In one embodiment, the 5′ PB1or PB2 incorporation sequences correspond to the 3′ most nucleotides,e.g., the 3′ 1 to 250 nucleotides, 1 to 200 nucleotides, nucleotides 1to 150, nucleotides 1 to 160, 1 to 170 or 1 to 190, or any integerbetween 1 and 250, of the C-terminal PB1 or PB2 coding region.

In one embodiment, the 3′ PA incorporation sequences correspond tonucleotides 1 to 250, nucleotides 1 to 200, nucleotides 1 to 150, or anyinteger between 1 and 250, of the N-terminal PA coding region. In oneembodiment, the 5′ PA incorporation sequences correspond to the 3′ mostnucleotides, e.g., the 3′ 1 to 250 nucleotides, 1 to 200 nucleotides,nucleotides 1 to 150, nucleotides 1 to 160, 1 to 170 or 1 to 190, or anyinteger between 1 and 250, of the C-terminal PA coding region.

In one embodiment, the 3′ M incorporation sequences correspond tonucleotides 1 to 250, nucleotides 1 to 242, nucleotides 1 to 240 or anyinteger between 1 and 250, of the N-terminal M coding region, and mayinclude a mutation at the M initiation codon. In another embodiment, the5′ M incorporation sequences correspond to sequences in the C-terminalcoding region of M, sequences corresponding to the 3′ most 50, 100, or220, or any integer between 1 and 250, nucleotides for C-terminal Mcoding region.

In one embodiment, the 3′ NS or NP incorporation sequences correspond tonucleotides 1 to 250, nucleotides 1 to 200, nucleotides 1 to 150,nucleotides 1 to 30, or any integer between 1 and 250, e.g., 1 to 60, 1to 70, 1 to 80 or 1 to 90 of the N-terminal NS or NP coding region, andmay include a mutation at the NS or NP initiation codon. In anotherembodiment, the 5′ NS or NP incorporation sequences correspond tosequences in the C-terminal coding region of NS or NP, sequencescorresponding to the 3′ most 10, 30, 150, 200 or 250, or any integerbetween 1 and 250, nucleotides for the C-terminal NS or NP codingregion, e.g., nucleotides 1 to 250, nucleotides 1 to 200, nucleotides 1to 150, nucleotides 1 to 30, or any integer between 1 and 250, e.g., 1to 60, 1 to 70, 1 to 80 or 1 to 90 of the C-terminal NS or NP codonregion.

Accordingly, the disclosure provides influenza virus vectors whichinclude sequences corresponding to the 3′ and 5′ noncoding regions of aparticular vRNA, incorporation sequences of the corresponding vRNA, anda heterologous nucleic acid segment. Thus, in one embodiment, the vectorincludes the 3′ noncoding region of NA vRNA, 3′ or 5′ NA vRNAincorporation sequences, and optionally both 3′ and 5′ NA incorporationsequences, a heterologous nucleic acid segment, and the 5′ noncodingregion of NA vRNA. In another embodiment, the vector includes the 3′noncoding region of HA vRNA, 5′ or 3′ HA vRNA incorporation sequences orboth 5′ and 3′ HA incorporation sequences, a heterologous nucleic acidsegment, and the 5′ noncoding region of HA vRNA. In another embodiment,the vector includes the 3′ noncoding region of NS vRNA, NS incorporationsequences, a heterologous nucleic acid segment, and the 5′ noncodingregion of NS vRNA. In another embodiment, the vector includes the 3′noncoding region of M vRNA, 5′ or 3′ M incorporation sequences or both5′ and 3′ M incorporation sequences, a heterologous nucleic acidsegment, and the 5′ noncoding region of M vRNA. In yet anotherembodiment, the vector includes the 3′ noncoding region of PB2 vRNA, aheterologous nucleic acid segment, PB2 incorporation sequences, and the5′ noncoding region of PB2 vRNA. When two incorporation sequences areemployed in a vector, they may be separated by the heterologous nucleicacid segment. Each vector may be employed so as to prepare vRNA forintroduction to a cell, or to express vRNA in a cell, in which otherinfluenza virus vRNAs and proteins necessary for virus production, arepresent.

In another embodiment, the heterologous gene sequence comprisessequences corresponding to an open reading frame for a therapeutic gene.In yet a further embodiment, the heterologous gene sequence comprisessequences corresponding to an open reading frame for an immunogenicpeptide or protein of a pathogen or a tumor cell, e.g., one useful toinduce a protective immune response. For example, the heterologousnucleic acid segment may encode an immunogenic epitope useful in cancertherapy or a vaccine. The vector comprising the heterologous nucleicacid segment may be prepared such that transcription of vector vRNAresults in mRNA encoding a fusion protein with an influenza protein suchas NA. Thus, it is envisioned that the heterologous nucleic acid segmentmay be fused with viral incorporation sequences so as to encode a fusionprotein, e.g., a fusion with the N-terminal 21 residues of NA. Thefusion protein may comprise sequences from two different influenza virusproteins including sequences from two different NA or HA proteins. Inanother embodiment, the heterologous nucleic acid segment may comprisesequences corresponding to an IRES linked 5N to an open reading frame.

In one embodiment of the disclosure, the heterologous gene sequence mayencode a heterologous protein (a non-influenza viral protein such as aglycoprotein or a cytosolic, nuclear or mitochondrial specific protein),which may confer a detectable phenotype. In one embodiment, theheterologous gene sequence may be fused to truncated portions of PB2coding sequences, e.g., those corresponding to 5′ or 3′ PB2 codingincorporation sequences, optionally forming a chimeric protein. In oneembodiment, the heterologous nucleotide sequence replaces or isintroduced to sequences in the viral viral segment corresponding to thecoding region for that segment, so as not to disrupt the incorporationsequences in the coding region of the viral segment. For instance, theheterologous nucleotide sequence may be flanked by about 3 to about 400nucleotides of the 5′ and/or 3′ PB2 coding region adjacent to non-codingsequence. In one embodiment, the 3′ PB2 incorporation sequencescorrespond to nucleotides 3 to 400, nucleotides 3 to 300, nucleotides 3to 100, nucleotides 3 to 50, or any integer between 3 and 400, of theN-terminal and/or C-terminal PB2 coding region. In one embodiment, afterinfection of a host cell with the biologically contained PB2-KO virus, aheterologous protein is produced which is a fusion with the N-terminusand/or C-terminus of the remaining residues of the deleted PB2 protein.

The vRNA for the additional viral segment or a viral segment having theheterologous gene sequence may be incorporated into virions at anefficiency that is at least 1%, 5%, 10%, or 30%, or at least 50%, thatof a corresponding wild-type vRNA.

Influenza Virus Structure and Propagation

Influenza A viruses possess a genome of eight single-strandednegative-sense viral RNAs (vRNAs) that encode at least ten proteins. Theinfluenza virus life cycle begins with binding of the hemagglutinin (HA)to sialic acid-containing receptors on the surface of the host cell,followed by receptor-mediated endocytosis. The low pH in late endosomestriggers a conformational shift in the HA, thereby exposing theN-terminus of the HA2 subunit (the so-called fusion peptide). The fusionpeptide initiates the fusion of the viral and endosomal membrane, andthe matrix protein (M1) and RNP complexes are released into thecytoplasm. RNPs consist of the nucleoprotein (NP), which encapsidatesvRNA, and the viral polymerase complex, which is formed by the PA, PB1,and PB2 proteins. RNPs are transported into the nucleus, wheretranscription and replication take place. The RNA polymerase complexcatalyzes three different reactions: synthesis of an mRNA with a 5′ capand 3′ polyA structure, of a full-length complementary RNA (cRNA), andof genomic vRNA using the cRNA as a template. Newly synthesized vRNAs,NP, and polymerase proteins are then assembled into RNPs, exported fromthe nucleus, and transported to the plasma membrane, where budding ofprogeny virus particles occurs. The neuraminidase (NA) protein plays acrucial role late in infection by removing sialic acid fromsialyloligosaccharides, thus releasing newly assembled virions from thecell surface and preventing the self-aggregation of virus particles.Although virus assembly involves protein-protein and protein-vRNAinteractions, the nature of these interactions is largely unknown.

Although influenza B and C viruses are structurally and functionallysimilar to influenza A virus, there are some differences. For example,influenza B virus does not have a M2 protein with ion channel activitybut has BM2 and has a viral segment with both NA and NB sequences.Influenza C virus has only seven viral segments.

Cell Lines that can be Used

Any cell, e.g., any avian or mammalian cell, such as a human, e.g., 293Tor PER.C69 cells, or canine, e.g., MDCK, bovine, equine, feline, swine,ovine, rodent, for instance mink, e.g., MvLu1 cells, or hamster, e.g.,CHO cells, or non-human primate, e.g., Vero cells, including mutantcells, which supports efficient replication of influenza virus can beemployed to isolate and/or propagate influenza viruses. Isolated virusescan be used to prepare a reassortant virus. In one embodiment, hostcells for vaccine production are continuous mammalian or avian celllines or cell strains. A complete characterization of the cells to beused, may be conducted so that appropriate tests for purity of the finalproduct can be included. Data that can be used for the characterizationof a cell includes (a) information on its origin, derivation, andpassage history; (b) information on its growth and morphologicalcharacteristics; (c) results of tests of adventitious agents; (d)distinguishing features, such as biochemical, immunological, andcytogenetic patterns which allow the cells to be clearly recognizedamong other cell lines; and (e) results of tests for tumorigenicity. Inone embodiment, the passage level, or population doubling, of the hostcell used is as low as possible.

In one embodiment, the cells are WHO certified, or certifiable,continuous cell lines. The requirements for certifying such cell linesinclude characterization with respect to at least one of genealogy,growth characteristics, immunological markers, virus susceptibilitytumorigenicity and storage conditions, as well as by testing in animals,eggs, and cell culture. Such characterization is used to confirm thatthe cells are free from detectable adventitious agents. In somecountries, karyology may also be required. In addition, tumorigenicitymay be tested in cells that are at the same passage level as those usedfor vaccine production. The virus may be purified by a process that hasbeen shown to give consistent results, before vaccine production (see,e.g., World Health Organization, 1982).

Virus produced by the host cell may be highly purified prior to vaccineor gene therapy formulation.

Generally, the purification procedures result in extensive removal ofcellular DNA and other cellular components, and adventitious agents.Procedures that extensively degrade or denature DNA may also be used.

Influenza Vaccines

A vaccine of the disclosure includes an isolated recombinant influenzavirus of the disclosure, and optionally one or more other isolatedviruses including other isolated influenza viruses, one or moreimmunogenic proteins or glycoproteins of one or more isolated influenzaviruses or one or more other pathogens, e.g., an immunogenic proteinfrom one or more bacteria, non-influenza viruses, yeast or fungi, orisolated nucleic acid encoding one or more viral proteins (e.g., DNAvaccines) including one or more immunogenic proteins of the isolatedinfluenza virus of the disclosure. In one embodiment, the influenzaviruses of the disclosure may be vaccine vectors for influenza virus orother pathogens.

A complete virion vaccine may be concentrated by ultrafiltration andthen purified by zonal centrifugation or by chromatography. Virusesother than the virus of the invention, such as those included in amultivalent vaccine, may be inactivated before or after purificationusing formalin or beta-propiolactone, for instance.

A subunit vaccine comprises purified glycoproteins. Such a vaccine maybe prepared as follows: using viral suspensions fragmented by treatmentwith detergent, the surface antigens are purified, byultracentrifugation for example. The subunit vaccines thus containmainly HA protein, and also NA. The detergent used may be cationicdetergent for example, such as hexadecyl trimethyl ammonium bromide(Bachmeyer, 1975), an anionic detergent such as ammonium deoxycholate(Laver & Webster, 1976); or a nonionic detergent such as thatcommercialized under the name TRITON X100. The hemagglutinin may also beisolated after treatment of the virions with a protease such asbromelin, and then purified. The subunit vaccine may be combined with anattenuated virus of the disclosure in a multivalent vaccine.

A split vaccine comprises virions which have been subjected to treatmentwith agents that dissolve lipids. A split vaccine can be prepared asfollows: an aqueous suspension of the purified virus obtained as above,inactivated or not, is treated, under stirring, by lipid solvents suchas ethyl ether or chloroform, associated with detergents. Thedissolution of the viral envelope lipids results in fragmentation of theviral particles. The aqueous phase is recuperated containing the splitvaccine, constituted mainly of hemagglutinin and neuraminidase withtheir original lipid environment removed, and the core or itsdegradation products. Then the residual infectious particles areinactivated if this has not already been done. The split vaccine may becombined with an attenuated virus of the disclosure in a multivalentvaccine.

Inactivated Vaccines. Inactivated influenza virus vaccines are providedby inactivating replicated virus using known methods, such as, but notlimited to, formalin or β-propiolactone treatment. Inactivated vaccinetypes that can be used in the invention can include whole-virus (WV)vaccines or subvirion (SV) (split) vaccines. The WV vaccine containsintact, inactivated virus, while the SV vaccine contains purified virusdisrupted with detergents that solubilize the lipid-containing viralenvelope, followed by chemical inactivation of residual virus.

In addition, vaccines that can be used include those containing theisolated HA and NA surface proteins, which are referred to as surfaceantigen or subunit vaccines.

Live Attenuated Virus Vaccines. Live, attenuated influenza virusvaccines, such as those including a recombinant virus of the disclosurecan be used for preventing or treating influenza virus infection.Attenuation may be achieved in a single step by transfer of attenuatedgenes from an attenuated donor virus to a replicated isolate orreassorted virus according to known methods. Since resistance toinfluenza A virus is mediated primarily by the development of an immuneresponse to the HA and/or NA glycoproteins, the genes coding for thesesurface antigens come from the reassorted viruses or clinical isolates.The attenuated genes are derived from an attenuated parent. In thisapproach, genes that confer attenuation generally do not code for the HAand NA glycoproteins.

Viruses (donor influenza viruses) are available that are capable ofreproducibly attenuating influenza viruses, e.g., a cold adapted (ca)donor virus can be used for attenuated vaccine production. See, forexample, Isakova-Sivall et al., 2014. Live, attenuated reassortant virusvaccines can be generated by mating the ca donor virus with a virulentreplicated virus. Reassortant progeny are then selected at 25° C.(restrictive for replication of virulent virus), in the presence of anappropriate antiserum, which inhibits replication of the viruses bearingthe surface antigens of the attenuated ca donor virus. Usefulreassortants are: (a) infectious, (b) attenuated for seronegativenon-adult mammals and immunologically primed adult mammals, (c)immunogenic and (d) genetically stable. The immunogenicity of the careassortants parallels their level of replication. Thus, the acquisitionof the six transferable genes of the ca donor virus by new wild-typeviruses has reproducibly attenuated these viruses for use in vaccinatingsusceptible mammals both adults and non-adult.

Other attenuating mutations can be introduced into influenza virus genesby site-directed mutagenesis to rescue infectious viruses bearing thesemutant genes. Attenuating mutations can be introduced into non-codingregions of the genome, as well as into coding regions. Such attenuatingmutations can also be introduced into genes other than the HA or NA,e.g., the PB2 polymerase gene. Thus, new donor viruses can also begenerated bearing attenuating mutations introduced by site-directedmutagenesis, and such new donor viruses can be used in the production oflive attenuated reassortants vaccine candidates in a manner analogous tothat described above for the ca donor virus. Similarly, other known andsuitable attenuated donor strains can be reassorted with influenza virusto obtain attenuated vaccines suitable for use in the vaccination ofmammals.

In one embodiment, such attenuated viruses maintain the genes from thevirus that encode antigenic determinants substantially similar to thoseof the original clinical isolates. This is because the purpose of theattenuated vaccine is to provide substantially the same antigenicity asthe original clinical isolate of the virus, while at the same timelacking pathogenicity to the degree that the vaccine causes minimalchance of inducing a serious disease condition in the vaccinated mammal.

The viruses in a multivalent vaccine can thus be attenuated orinactivated, formulated and administered, according to known methods, asa vaccine to induce an immune response in an animal, e.g., a mammal.Methods are well-known in the art for determining whether suchattenuated or inactivated vaccines have maintained similar antigenicityto that of the clinical isolate or high growth strain derived therefrom.Such known methods include the use of antisera or antibodies toeliminate viruses expressing antigenic determinants of the donor virus;chemical selection (e.g., amantadine or rimantidine); HA and NA activityand inhibition; and nucleic acid screening (such as probe hybridizationor PCR) to confirm that donor genes encoding the antigenic determinants(e.g., HA or NA genes) are not present in the attenuated viruses.

Exemplary Embodiments

A reporter influenza virus, e.g., allowing visualization ofvirus-infected cells to understand influenza virus-induced pathology,was prepared by inserting the gene for the Venus fluorescent proteininto the NS segment of influenza A/Puerto Rico/8/34 (PR8, H1N1) virus toyield WT−Venus−PR8. Although the inserted Venus gene was deleted duringserial passages of WT−Venus−PR8, and WT−Venus−PR8 was significantlyattenuated, the PB2−E712D mutation was found to stabilize the Venusgene. As disclosed herein, the mechanisms by which Venus gene deletionoccurs and how the polymerase mutation stabilizes the Venus gene wereinvestigated. Deep sequencing analysis revealed that PB2−E712D does notcause an appreciable change in the mutation rate, suggesting that thestability of the Venus gene is not affected by polymerase fidelity.Using quantitative real-time PCR it was found that WT−Venus−PR8 induceshigh-level interferon beta (IFN-β) expression. The induction of IFN-βexpression seemed to result from the reduced transcription/replicationefficiency of the modified NS segment in WT−Venus−PR8. In contrast, thetranscription/replication efficiency of the modified NS segment wasenhanced by the PB2−E712D mutation. Loss of the Venus gene inWT−Venus−PR8 appeared to be caused by internal deletions in the NSsegment. Moreover, to further the understanding of the Venusstabilization mechanisms, additional amino acid mutations in the viruspolymerase complex were identified that stabilize the Venus gene. It wasfound that some of these amino acids are located near the template exitor the product exit of the viral polymerase, suggesting that these aminoacids contribute to the stability of the Venus gene by affecting thebinding affinity between the polymerase complex and the RNA template andproduct.

The disclosure provides an isolated recombinant influenza virus havingPA, PB1, PB2, NP, NS, M, NA, and HA viral segments, wherein at least oneof the viral segments is a PB2 viral segment encoding PB2 with residueat position 540 that is not asparagine, a PA viral segment encoding PAwith a residue at position 180 that is not glutamine or a residue atposition 200 that is not threonine, or a PB1 viral segment encoding PB1with a residue at position 149 that is not valine, a residue at position684 that is not glutamic acid or a residue at position 685 that is notaspartic acid, or any combination thereof, wherein the recombinantinfluenza virus has enhanced genetic stability or enhanced replicationrelative to a corresponding recombinant influenza virus with a residueat position 540 in PB2 that is asparagine, a residue at position 180 inPA that is glutamine, a residue at position 200 in PA that is threonine,a residue at position 149 in PB1 that is valine, a residue at position684 in PB1 that is glutamic acid or a residue at position 685 in PB1that is aspartic acid. In one embodiment, the residue at position 540 ofPB2 is K, R, D, E, Q, or H, the residue at position 712 of PB2 is D, N,S, H, T, Y, or C, the residue at position 180 in PA is R, K, D, E, N, orH, the residue at position 200 in PA is A, I, L, C, S. M, F, P, G, or V,the residue at position 149 in PB1 is A, T, I, L, C, S, M, F, P, or G,the residue at position 684 is D, Q, S, H, T, Y. C, K, R, or N, or theresidue at position 685 in PB1 is E, N, R, H, K, S, T, Y, C, or Q. Inone embodiment, the residue at position 540 of PB2 is K, R, H, D, S. H,T, Y, or C, the residue at position 712 of PB2 is D, K, H, R, Q, or N,the residue at position 180 in PA is R, K, D, N, S. H, T, Y, or H, theresidue at position 200 in PA is A, I, L, G, S, M, or V, the residue atposition 149 in PB1 is A, T, I, L, S, M, or G, the residue at position684 is D, Q, H, L, R or N, or the residue at position 685 in PB1 is E,N, R, H, K or Q. In one embodiment, the residue at position 540 of PB2is K, R or H, the residue at position 712 of PB2 is D or N, the residueat position 180 in PA is R, K or H, the residue at position 200 in PA isA, I, L, G or V, the residue at position 149 in PB1 is A, T, I, L or G,the residue at position 684 is D or N, or the residue at position 685 inPB1 is E or Q. In one embodiment, the PA further comprises a residue atposition 443 that is not arginine, the PB1 further comprises a residueat position 737 that is not lysine, the PB2 further comprises a residueat position 25 that is not valine or a residue at position 712 that isnot glutamic acid, the NS viral segment encodes a NS1 with a residue atposition 167 that is not proline, the HA viral segment encodes a HA witha residue at position 380 that is not threonine, or any combinationthereof. In one embodiment, the residue at position 443 of PA is K or H,the residue at position 737 of PB1 is H or R, the residue at position 25of PB2 is A, L, T, I, or G, the residue at position 712 of PB2 is D, theresidue at position 167 of NS1 is S, C, M, A, L, I, G or T, or anycombination thereof. In one embodiment, at least one of the viralsegments includes a heterologous gene sequence encoding a gene product.In one embodiment, the heterologous sequence is in the NS viral segment,M viral segment, NP viral segment, PA viral segment, PB1 viral segment,or the PB2 viral segment. In one embodiment, the heterologous sequenceis 5′ or 3′ to the PA coding sequence in the PA viral segment, 5′ or 3′to the PB1 coding sequence in the PB1 viral segment. In one embodiment,the heterologous sequence is 5′ or 3′ to the PB2 coding sequence in thePB2 viral segment. In one embodiment, the heterologous sequence is 5′ or3′ to the NS1 coding sequence in the NS viral segment. In oneembodiment, the recombinant virus comprises a further viral segmentcomprising a heterologous gene sequence encoding a gene product. In oneembodiment, the further viral segment is a NS viral segment, a M viralsegment, a NP viral segment, a PA viral segment, a PB1 viral segment ora PB2 viral segment. In one embodiment, the virus has a HA that is H1,H2, H3, H5, H7, H9, or H10. In one embodiment, the virus is an influenzaB virus.

Also provided is an isolated recombinant influenza virus having PA, PB1,PB2, NP, NS, M, NA, and HA viral segments, wherein at least one of theviral segments is a PB2 viral segment encoding PB2 with residue atposition 540 that is not asparagine or a residue at position 712 that isnot glutamic acid, and wherein at least one of the other viral segmentsis a PA viral segment encoding PA with a residue at position 180 that isnot glutamine or a residue at position 200 that is not threonine, or aPB1 viral segment encoding PB1 with a residue at position 149 that isnot valine, a residue at position 684 that is not glutamic acid or aresidue at position 685 that is not aspartic acid, or any combinationthereof, wherein the recombinant influenza virus has enhanced geneticstability or replication relative to a corresponding recombinantinfluenza virus with a residue at position 540 in PB2 that isasparagine, a residue in PB2 at position 712 that is glutamic acid, aresidue at position 180 in PA that is glutamine, a residue at position200 in PA that is threonine, a residue at position 149 in PB1 that isvaline, a residue at position 684 in PB1 that is glutamic acid or aresidue at position 685 in PB1 that is aspartic acid. In one embodiment,the residue at position 540 of PB2 is K, R, D, E, Q, or H, the residueat position 712 of PB2 is D, N, S, H, T, Y, or C, the residue atposition 180 in PA is R, K, D, E, N, or H, the residue at position 200in PA is A, I, L, C, S, M, F, P, G, or V, the residue at position 149 inPB1 is A, T, I, L, C, S, M, F, P, or G, the residue at position 684 isD, Q, S, H, T, Y, C, K, R, or N, or the residue at position 685 in PB1is E, N, R, H, K, S, T, Y, C, or Q. In one embodiment, the residue atposition 540 of PB2 is K, R, H, D, S, H, T, Y, or C, the residue atposition 712 of PB2 is D, K, H, R, Q, or N, the residue at position 180in PA is R, K, D, N, S, H, T, Y, or H, the residue at position 200 in PAis A, I, L, G, S, M, or V, the residue at position 149 in PB1 is A, T,I, L, S, M, or G, the residue at position 684 is D, Q, H, L, R or N, orthe residue at position 685 in PB1 is E, N, R, H, K or Q. In oneembodiment, the residue at position 540 of PB2 is K, R or H, the residueat position 712 of PB2 is D or N, the residue at position 180 in PA isR, K or H, the residue at position 200 in PA is A, I, L, G or V, theresidue at position 149 in PB1 is A, T, I, L or G, the residue atposition 684 is D or N, or the residue at position 685 in PB1 is E or Q.In one embodiment, the PA further comprises a residue at position 443that is not arginine, the PB1 further comprises a residue at position737 that is not lysine, the PB2 further comprises a residue at position25 that is not valine or a residue at position 712 that is not glutamicacid, the NS viral segment encodes a NS1 with a residue at position 167that is not proline, the HA viral segment encodes a HA with a residue atposition 380 that is not threonine, or any combination thereof. In oneembodiment, the residue at position 443 of PA is K or H, the residue atposition 737 of PB1 is H or R, the residue at position 25 of PB2 is A,L, T, I, or G, the residue at position 712 of PB2 is D, the residue atposition 167 of NS1 is S, C, M, A, L, I, G or T, or any combinationthereof. In one embodiment, at least one of the viral segments includesa heterologous gene sequence encoding a gene product. In one embodiment,the heterologous sequence is in the NS viral segment, M viral segment,NP viral segment, PA viral segment, PB1 viral segment, or the PB2 viralsegment. In one embodiment, the heterologous sequence is 5′ or 3′ to thePA coding sequence in the PA viral segment, 5′ or 3′ to the PB1 codingsequence in the PB1 viral segment. In one embodiment, the heterologoussequence is 5′ or 3′ to the PB2 coding sequence in the PB2 viralsegment. In one embodiment, the heterologous sequence is 5′ or 3′ to theNS1 coding sequence in the NS viral segment. In one embodiment, therecombinant virus comprises a further viral segment comprising aheterologous gene sequence encoding a gene product. In one embodiment,the further viral segment is a NS viral segment, a M viral segment, a NPviral segment, a PA viral segment, a PB1 viral segment or a PB2 viralsegment. In one embodiment, the virus has a HA that is H1, H2, H3, H5,H7, H9, or H10. In one embodiment, the virus is an influenza B virus.

Further provided is an isolated recombinant influenza virus having PA,PB1, PB2, NP, NS, M, NA, and HA viral segments, wherein the recombinantvirus has two or more viral segments comprising a PB2 viral segmentencoding PB2 with residue at position 540 that is not asparagine or aresidue at position 712 that is not glutamic acid, a PA viral segmentencoding PA with a residue at position 180 that is not glutamine or aresidue at position 200 that is not threonine, or a PB1 viral segmentencoding PB1 with a residue at position 149 that is not valine, aresidue at position 684 that is not glutamic acid or a residue atposition 685 that is not aspartic acid, or any combination thereof,wherein the recombinant influenza virus has enhanced genetic stabilityor replication relative to a corresponding recombinant influenza viruswith a residue at position 540 in PB2 that is asparagine, a residue inPB2 at position 712 that is glutamic acid, a residue at position 180 inPA that is glutamine, a residue at position 200 in PA that is threonine,a residue at position 149 in PB1 that is valine, a residue at position684 in PB1 that is glutamic acid or a residue at position 685 in PB1that is aspartic acid. In one embodiment, the residue at position 540 ofPB2 is K, R, D, E, Q, or H, the residue at position 712 of PB2 is D, N,S, H, T, Y, or C, the residue at position 180 in PA is R, K, D, E, N, orH, the residue at position 200 in PA is A, I, L, C, S, M, F, P, G, or V,the residue at position 149 in PB1 is A, T, I, L, C, S, M, F, P, or G,the residue at position 684 is D, Q, S, H, T, Y, C, K, R, or N, or theresidue at position 685 in PB1 is E, N, R, H, K, S, T, Y, C, or Q. Inone embodiment, the residue at position 540 of PB2 is K, R, H, D, S, H,T, Y, or C, the residue at position 712 of PB2 is D, K, H, R, Q, or N,the residue at position 180 in PA is R, K, D, N, S, H, T, Y, or H, theresidue at position 200 in PA is A, I, L, G, S, M, or V, the residue atposition 149 in PB1 is A, T, I, L, S, M, or G, the residue at position684 is D, Q, H, L, R or N, or the residue at position 685 in PB1 is E,N, R, H, K or Q. In one embodiment, the residue at position 540 of PB2is K, R or H, the residue at position 712 of PB2 is D or N, the residueat position 180 in PA is R, K or H, the residue at position 200 in PA isA, I, L, G or V, the residue at position 149 in PB1 is A, T, I, L or G,the residue at position 684 is D or N, or the residue at position 685 inPB1 is E or Q. In one embodiment, the PA further comprises a residue atposition 443 that is not arginine, the PB1 further comprises a residueat position 737 that is not lysine, the PB2 further comprises a residueat position 25 that is not valine or a residue at position 712 that isnot glutamic acid, the NS viral segment encodes a NS1 with a residue atposition 167 that is not proline, the HA viral segment encodes a HA witha residue at position 380 that is not threonine, or any combinationthereof. In one embodiment, the residue at position 443 of PA is K or H,the residue at position 737 of PB1 is H or R, the residue at position 25of PB2 is A, L, T, I, or G, the residue at position 712 of PB2 is D, theresidue at position 167 of NS1 is S, C, M, A, L, I, G or T, or anycombination thereof. In one embodiment, at least one of the viralsegments includes a heterologous gene sequence encoding a gene product.In one embodiment, the heterologous sequence is in the NS viral segment,M viral segment, NP viral segment, PA viral segment, PB1 viral segment,or the PB2 viral segment. In one embodiment, the heterologous sequenceis 5′ or 3′ to the PA coding sequence in the PA viral segment, 5′ or 3′to the PB1 coding sequence in the PB1 viral segment. In one embodiment,the heterologous sequence is 5′ or 3′ to the PB2 coding sequence in thePB2 viral segment. In one embodiment, the heterologous sequence is 5′ or3′ to the NS1 coding sequence in the NS viral segment. In oneembodiment, the recombinant virus comprises a further viral segmentcomprising a heterologous gene sequence encoding a gene product. In oneembodiment, the further viral segment is a NS viral segment, a M viralsegment, a NP viral segment, a PA viral segment, a PB1 viral segment ora PB2 viral segment. In one embodiment, the virus has a HA that is H1,H2, H3, H5, H7, H9, or H10. In one embodiment, the virus is an influenzaB virus.

The disclosure also provides a vaccine having the isolated recombinantvirus.

The disclosure provides a plurality of influenza virus vectors forpreparing a reassortant, comprising a vector for vRNA productioncomprising a promoter operably linked to an influenza virus PA DNAlinked to a transcription termination sequence, a vector for vRNAproduction comprising a promoter operably linked to an influenza virusPB1 DNA linked to a transcription termination sequence, a vector forvRNA production comprising a promoter operably linked to an influenzavirus PB2 DNA linked to a transcription termination sequence, a vectorfor vRNA production comprising a promoter operably linked to aninfluenza virus HA DNA linked to a transcription termination sequence, avector for vRNA production comprising a promoter operably linked to aninfluenza virus NP DNA linked to a transcription termination sequence, avector for vRNA production comprising a promoter operably linked to aninfluenza virus NA DNA linked to a transcription termination sequence, avector for vRNA production comprising a promoter operably linked to aninfluenza virus M DNA linked to a transcription termination sequence,and a vector for vRNA production comprising a promoter operably linkedto an influenza virus NS cDNA linked to a transcription terminationsequence, wherein the PB1, PB2, or PA DNAs in the vectors for vRNAproduction encode at least one of: a PB2 viral segment encoding PB2 withresidue at position 540 that is not asparagine, a PA viral segmentencoding PA with a residue at position 180 that is not glutamine or aresidue at position 200 that is not threonine, or a PB1 viral segmentencoding PB1 with a residue at position 149 that is not valine, aresidue at position 684 that is not glutamic acid or a residue atposition 685 that is not aspartic acid, or a combination thereof; andoptionally a vector for mRNA production comprising a promoter operablylinked to a DNA segment encoding influenza virus PA, a vector for mRNAproduction comprising a promoter operably linked to a DNA segmentencoding influenza virus PB1, a vector for mRNA production comprising apromoter operably linked to a DNA segment encoding influenza virus PB2,and a vector for mRNA production comprising a promoter operably linkedto a DNA segment encoding influenza virus NP, and optionally a vectorfor mRNA production comprising a promoter operably linked to a DNAsegment encoding influenza virus HA, a vector for mRNA productioncomprising a promoter operably linked to a DNA segment encodinginfluenza virus NA, a vector for mRNA production comprising a promoteroperably linked to a DNA segment encoding influenza virus M1, a vectorfor mRNA production comprising a promoter operably linked to a DNAsegment encoding influenza virus M2, or a vector for mRNA productioncomprising a promoter operably linked to a DNA segment encodinginfluenza virus NS2. In one embodiment, the PB1, PB2, PA, NP, NS, and MDNAs in the vectors for vRNA production have a sequence corresponding toone that encodes a polypeptide having at least 95% amino acid sequenceidentity to a corresponding polypeptide encoded by SEQ ID NOs:1-6 or10-15. In one embodiment, the residue at position 540 of PB2 is K, R orH, the residue at position 180 in PA is R, K or H, the residue atposition 200 in PA is A, I, L, G or V, the residue at position 149 inPB1 is A, T, I, L or G, the residue at position 684 is D or N, or theresidue at position 685 in PB1 is E or Q. In one embodiment, at leastone of the viral segments includes a heterologous gene sequence encodinga gene product. In one embodiment, the vectors comprise a further vectorhaving a viral segment comprising a heterologous gene sequence encodinga gene product.

A method to prepare influenza virus is provided, comprising: contactinga cell with a vector for vRNA production comprising a promoter operablylinked to an influenza virus PA DNA linked to a transcriptiontermination sequence, a vector for vRNA production comprising a promoteroperably linked to an influenza virus PB1 DNA linked to a transcriptiontermination sequence, a vector for vRNA production comprising a promoteroperably linked to an influenza virus PB2 DNA linked to a transcriptiontermination sequence, a vector for vRNA production comprising a promoteroperably linked to an influenza virus HA DNA linked to a transcriptiontermination sequence, a vector for vRNA production comprising a promoteroperably linked to an influenza virus NP DNA linked to a transcriptiontermination sequence, a vector for vRNA production comprising a promoteroperably linked to an influenza virus NA DNA linked to a transcriptiontermination sequence, a vector for vRNA production comprising a promoteroperably linked to an influenza virus M DNA linked to a transcriptiontermination sequence, and a vector for vRNA production comprising apromoter operably linked to an influenza virus NS DNA linked to atranscription termination sequence, wherein the PB1, PB2, or PA DNAs inthe vectors for vRNA production encode i) a PB2 with residue at position540 that is not asparagine or a residue at position 712 that is notglutamic acid, and at least one: a PA with a residue at position 180that is not glutamine or a residue at position 200 that is notthreonine, or a PB1 with a residue at position 149 that is not valine, aresidue at position 684 that is not glutamic acid or a residue atposition 685 that is not aspartic acid, or any combination thereof, ii)a PB2 with residue at position 540 that is not asparagine, a PA with aresidue at position 180 that is not glutamine or a residue at position200 that is not threonine, or a PB1 with a residue at position 149 thatis not valine, a residue at position 684 that is not glutamic acid or aresidue at position 685 that is not aspartic acid, or any combinationthereof, or iii) two or more of: a PB2 with residue at position 540 thatis not asparagine or a residue at position 712 that is not glutamicacid, a PA with a residue at position 180 that is not glutamine or aresidue at position 200 that is not threonine, or a PB1 with a residueat position 149 that is not valine, a residue at position 684 that isnot glutamic acid or a residue at position 685 that is not asparticacid, or any combination thereof; and optionally a vector for mRNAproduction comprising a promoter operably linked to a DNA segmentencoding influenza virus PA, a vector for mRNA production comprising apromoter operably linked to a DNA segment encoding influenza virus PB1,a vector for mRNA production comprising a promoter operably linked to aDNA segment encoding influenza virus PB2, and a vector for mRNAproduction comprising a promoter operably linked to a DNA segmentencoding influenza virus NP, and optionally a vector for mRNA productioncomprising a promoter operably linked to a DNA segment encodinginfluenza virus HA, a vector for mRNA production comprising a promoteroperably linked to a DNA segment encoding influenza virus NA, a vectorfor mRNA production comprising a promoter operably linked to a DNAsegment encoding influenza virus M1, a vector for mRNA productioncomprising a promoter operably linked to a DNA segment encodinginfluenza virus M2, or a vector for mRNA production comprising apromoter operably linked to a DNA segment encoding influenza virus NS2;in an amount effective to yield infectious influenza virus. In oneembodiment, the cell is an avian cell or a mammalian cell. In oneembodiment, the cell is a Vero cell, a human cell or a MDCK cell. In oneembodiment, the wherein the PB1, PB2, PA, NP, NS, and M DNAs in thevectors for vRNA productions have a sequence that corresponds to onethat encodes a polypeptide having at least 95% amino acid sequenceidentity to a corresponding polypeptide encoded by SEQ ID NOs:1-6 or10-15. In one embodiment, the residue at position 540 of PB2 is K, R orH, the residue at position 712 of PB2 is D or N, the residue at position180 in PA is R, K or H, the residue at position 200 in PA is A, I, L, Gor V, the residue at position 149 in PB1 is A, T, I, L or G, the residueat position 684 is D or N, or the residue at position 685 in PB1 is E orQ.

The heterologous sequence, e.g., for a therapeutic or prophylactic geneof interest, which may be in an additional influenza segment, e.g., inone of the segments of a 9 segment influenza A or B virus, in one of 8viral segments, or in one of the segments in a 7 segment virus, may bean immunogen for a cancer associated antigen or for a pathogen such as abacteria, a noninfluenza virus, fungus. In one embodiment, the influenzaviruses of the disclosure may be vaccine vectors for influenza virus andfor at least one other pathogen, such as a viral or bacterial pathogen,or for a pathogen other than influenza virus, pathogens including butnot limited to, lentiviruses such as HIV, hepatitis B virus, hepatitis Cvirus, herpes viruses such as CMV or HSV, Foot and Mouth Disease Virus,Measles virus, Rubella virus, Mumps virus, human Rhinovirus,Parainfluenza viruses, such as respiratory syncytial virus and humanparainfluenza virus type 1, Coronavirus, Nipah virus, Hantavirus,Japanese encephalitis virus, Rotavirus, Dengue virus, West Nile virus,Streptococcus pneumoniae, Mycobacterium tuberculosis, Bordetellapertussis, or Haemophilus influenza. For example, the biologicallycontained influenza virus of the disclosure may include sequences for Hprotein of Measles virus, viral envelope protein E1 of Rubella virus, HNprotein of Mumps virus, RV capsid protein VP1 of human Rhinovirus, Gprotein of Respiratory syncytial virus, S protein of Coronavirus, G or Fprotein of Nipah virus, G protein of Hantavirus, E protein of Japaneseencephalitis virus, VP6 of Rotavirus, E protein of Dengue virus, Eprotein of West Nile virus, PspA of Streptococcus pneumonia, HSP65 fromMycobacterium tuberculosis, IRP1-3 of Bordetella pertussis, or the hemeutilization protein, protective surface antigen D15, heme bindingprotein A, or outer membrane protein P1, P2, P5 or P6 of Haemophilusinfluenza. The gene therapy vector may include a heterologous sequenceuseful to inhibit or treat, e.g., cancer, AIDS, adenosine deaminase,muscular dystrophy, ornithine transcarbamylase deficiency and centralnervous system tumors, or pathogens, or may encode an antibody orfragment thereof, e.g., scFv or a single chain antibody.

Pharmaceutical Compositions

Pharmaceutical compositions of the present disclosure, suitable forinoculation, e.g., nasal, parenteral or oral administration, compriseone or more influenza virus isolates, e.g., one or more attenuated orinactivated influenza viruses, a subunit thereof, isolated protein(s)thereof, and/or isolated nucleic acid encoding one or more proteinsthereof, optionally further comprising sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. The compositions can furthercomprise auxiliary agents or excipients, as known in the art. Thecomposition of the disclosure is generally presented in the form ofindividual doses (unit doses).

Conventional vaccines generally contain about 0.1 to 200 μg, e.g., 30 to100 μg, 0.1 to 2 μg, 0.5 to 5 μg, 1 to 10 μg, 10 μg to 20 μg, 15 μg to30 μg, or 10 to 30 μg, of HA from each of the strains entering intotheir composition. The vaccine forming the main constituent of thevaccine composition of the disclosure may comprise a single influenzavirus, or a combination of influenza viruses, for example, at least twoor three influenza viruses, including one or more reassortant(s).

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and/or emulsions, which may containauxiliary agents or excipients known in the art. Examples of non-aqueoussolvents are propylene glycol, polyethylene glycol, vegetable oils suchas olive oil, and injectable organic esters such as ethyl oleate.Carriers or occlusive dressings can be used to increase skinpermeability and enhance antigen absorption. Liquid dosage forms fororal administration may generally comprise a liposome solutioncontaining the liquid dosage form. Suitable forms for suspendingliposomes include emulsions, suspensions, solutions, syrups, and elixirscontaining inert diluents commonly used in the art, such as purifiedwater. Besides the inert diluents, such compositions can also includeadjuvants, wetting agents, emulsifying and suspending agents, orsweetening, flavoring, or perfuming agents.

When a composition of the present disclosure is used for administrationto an individual, it can further comprise salts, buffers, adjuvants, orother substances which are desirable for improving the efficacy of thecomposition. For vaccines, adjuvants, substances which can augment aspecific immune response, can be used. Normally, the adjuvant and thecomposition are mixed prior to presentation to the immune system, orpresented separately, but into the same site of the organism beingimmunized.

Heterogeneity in a vaccine may be provided by mixing replicatedinfluenza viruses for at least two influenza virus strains, such as 2-20strains or any range or value therein. Vaccines can be provided forvariations in a single strain of an influenza virus, using techniquesknown in the art.

A pharmaceutical composition according to the present disclosure mayfurther or additionally comprise at least one chemotherapeutic compound,for example, for gene therapy, immunosuppressants, anti-inflammatoryagents or immune enhancers, and for vaccines, chemotherapeuticsincluding, but not limited to, gamma globulin, amantadine, guanidine,hydroxybenzimidazole, interferon-α, interferon-β, interferon-γ, tumornecrosis factor-alpha, thiosemicarbarzones, methisazone, rifampin,ribavirin, a pyrimidine analog, a purine analog, foscarnet,phosphonoacetic acid, acyclovir, dideoxynucleosides, a proteaseinhibitor, or ganciclovir.

The composition can also contain variable but small quantities ofendotoxin-free formaldehyde, and preservatives, which have been foundsafe and not contributing to undesirable effects in the organism towhich the composition is administered.

Pharmaceutical Purposes

The administration of the composition (or the antisera that it elicits)may be for either a “prophylactic” or “therapeutic” purpose. Whenprovided prophylactically, the compositions of the disclosure which arevaccines are provided before any symptom or clinical sign of a pathogeninfection becomes manifest. The prophylactic administration of thecomposition serves to prevent or attenuate any subsequent infection.When provided prophylactically, the gene therapy compositions of thedisclosure, are provided before any symptom or clinical sign of adisease becomes manifest. The prophylactic administration of thecomposition serves to prevent or attenuate one or more symptoms orclinical signs associated with the disease.

When provided therapeutically, a viral vaccine is provided upon thedetection of a symptom or clinical sign of actual infection. Thetherapeutic administration of the compound(s) serves to attenuate anyactual infection. When provided therapeutically, a gene therapycomposition is provided upon the detection of a symptom or clinical signof the disease. The therapeutic administration of the compound(s) servesto attenuate a symptom or clinical sign of that disease.

Thus, a vaccine composition of the present disclosure may be providedeither before the onset of infection (so as to prevent or attenuate ananticipated infection) or after the initiation of an actual infection.Similarly, for gene therapy, the composition may be provided before anysymptom or clinical sign of a disorder or disease is manifested or afterone or more symptoms are detected.

A composition is said to be “pharmacologically acceptable” if itsadministration can be tolerated by a recipient mammal. Such an agent issaid to be administered in a “therapeutically effective amount” if theamount administered is physiologically significant. A composition of thepresent disclosure is physiologically significant if its presenceresults in a detectable change in the physiology of a recipient patient,e.g., enhances at least one primary or secondary humoral or cellularimmune response against at least one strain of an infectious influenzavirus.

The “protection” provided need not be absolute, i.e., the influenzainfection need not be totally prevented or eradicated, if there is astatistically significant improvement compared with a control populationor set of mammals. Protection may be limited to mitigating the severityor rapidity of onset of symptoms or clinical signs of the influenzavirus infection.

Pharmaceutical Administration

A composition of the present disclosure may confer resistance to one ormore pathogens, e.g., one or more influenza virus strains, by eitherpassive immunization or active immunization. In active immunization, anattenuated live vaccine composition is administered prophylactically toa host (e.g., a mammal), and the host's immune response to theadministration protects against infection and/or disease. For passiveimmunization, the elicited antisera can be recovered and administered toa recipient suspected of having an infection caused by at least oneinfluenza virus strain. A gene therapy composition of the presentdisclosure may yield prophylactic or therapeutic levels of the desiredgene product by active immunization.

In one embodiment, the vaccine is provided to a mammalian female (at orprior to pregnancy or parturition), under conditions of time and amountsufficient to cause the production of an immune response which serves toprotect both the female and the fetus or newborn (via passiveincorporation of the antibodies across the placenta or in the mother'smilk).

The present disclosure thus includes methods for preventing orattenuating a disorder or disease, e.g., an infection by at least onestrain of pathogen. As used herein, a vaccine is said to prevent orattenuate a disease if its administration results either in the total orpartial attenuation (i.e., suppression) of a clinical sign or conditionof the disease, or in the total or partial immunity of the individual tothe disease. As used herein, a gene therapy composition is said toprevent or attenuate a disease if its administration results either inthe total or partial attenuation (i.e., suppression) of a clinical signor condition of the disease, or in the total or partial immunity of theindividual to the disease.

A composition having at least one influenza virus of the presentdisclosure, including one which is attenuated and one or more otherisolated viruses, one or more isolated viral proteins thereof, one ormore isolated nucleic acid molecules encoding one or more viral proteinsthereof, or a combination thereof, may be administered by any means thatachieve the intended purposes.

For example, administration of such a composition may be by variousparenteral routes such as subcutaneous, intravenous, intradermal,intramuscular, intraperitoneal, intranasal, oral or transdermal routes.

Parenteral administration can be accomplished by bolus injection or bygradual perfusion over time.

A typical regimen for preventing, suppressing, or treating an influenzavirus related pathology, comprises administration of an effective amountof a vaccine composition as described herein, administered as a singletreatment, or repeated as enhancing or booster dosages, over a period upto and including between one week and about 24 months, or any range orvalue therein.

According to the present disclosure, an “effective amount” of acomposition is one that is sufficient to achieve a desired effect. It isunderstood that the effective dosage may be dependent upon the species,age, sex, health, and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment, and the nature of the effectwanted. The ranges of effective doses provided below are not intended tolimit the invention and represent dose ranges.

The dosage of a live, attenuated or killed virus vaccine for an animalsuch as a mammalian adult organism may be from about 10²-10²⁰, e.g.,10³-10¹², 10²-10¹⁰, 10⁵-10¹¹, 10⁶-10¹⁵, 10²-10¹⁰, or 10¹⁵-10²⁰ plaqueforming units (PFU)/kg, or any range or value therein. The dose of oneviral isolate vaccine, e.g., in an inactivated vaccine, may range fromabout 0.1 to 1000, e.g., 0.1 to 10 μg, 1 to 20 μg, 30 to 100 μg, 10 to50 μg, 50 to 200 μg, or 150 to 300 μg, of HA protein. However, thedosage should be a safe and effective amount as determined byconventional methods, using existing vaccines as a starting point.

The dosage of immunoreactive HA in each dose of replicated virus vaccinemay be standardized to contain a suitable amount, e.g., 0.1 μg to 1 μg,0.5 μg to 5 μg, 1 μg to 10 μg, 10 μg to 20 μg, 15 μg to 30 μg, or 30 μgto 100 μg or any range or value therein, or the amount recommended bygovernment agencies or recognized professional organizations. Thequantity of NA can also be standardized, however, this glycoprotein maybe labile during purification and storage.

The dosage of immunoreactive HA in each dose of replicated virus vaccinecan be standardized to contain a suitable amount, e.g., 1-50 μg or anyrange or value therein, or the amount recommended by the U.S. PublicHealth Service (PHS), which is usually 15 μg, per component for olderchildren >3 years of age, and 7.5 μg per component for children <3 yearsof age. The quantity of NA can also be standardized, however, thisglycoprotein can be labile during the processor purification and storage(Kendal et al., 1980; Kerr et al., 1975). Each 0.5-ml dose of vaccinemay contain approximately 0.1 to 0.5 billion viral particles, 0.5 to 2billion viral particles, 1 to 50 billion virus particles, 1 to 10billion viral particles, 20 to 40 billion viral particles, 1 to 5billion viral particles, or 40 to 80 billion viral particles.

The invention will be further described by the following non-limitingexamples.

Example I Methods Generation of Color-Flu

The NS segments of PR8 fused with different fluorescent reporter genesincluding eCFP, eGFP, Venus, and mCherry were constructed by overlappingfusion PCR as described in Manicassamy et al. (2010). In brief, the openreading frame (ORF) of the NS1 gene without the stop codon was fusedwith the N-terminus of fluorescent reporter genes via a sequenceencoding the amino acid linker GSGG. The fluorescent reporter ORFs werefollowed by a sequence encoding the GSG linker, a foot-and-mouth virusprotease 2A autoproteolytic site with 57 nucleotides from porcineteschovirus-1 in Manicassamy et al. (2010), and by the ORF of nuclearexport protein (NEP) (FIG. 5). In addition, silent mutations wereintroduced into the endogenous splice acceptor site of the NS1 gene toabrogate splicing (Basler et al., 2001). The constructed NS segments(designated eCFP-NS, eGFP-NS, Venus-NS, and mCherry-NS) weresubsequently cloned into a pPoll vector for reverse genetics asdescribed in Newmann et al. (1999). The plasmid encoding the Venusreporter protein was a kind gift from Dr. A. Miyawaki (Laboratory forCell Function Dynamics, RIKEN Brain Science Institute, Wako, Japan)(Nagai et al., 2002). WT−Venus−PR8 was generated by using the reversegenetics system as described in Newmann et al. (1999). SinceWT−Venus−PR8 pathogenicity and Venus expression levels were appreciablyattenuated in mice, WT−Venus−PR8 was serially passaged in mice. Aftersix passages, a variant (MA−Venus−PR8) was obtained with increasedpathogenicity and strong Venus expression. A stock of MA−Venus−PR8 wasgenerated in MDCK cells. Since serial passage in animals typicallyresults in virus populations composed of genetic variants, MA−Venus−PR8was recreated by using reverse genetics. Likewise, MA−eCFP-PR8,−eGFP-PR8, and −mCherry-PR8 were generated with the same geneticbackbone as MA−Venus−PR8.

To generate a Venus−HPAI virus by reverse genetics, the NS segment ofA/Vietnam/1203/2004 (H5N1; VN1203) was replaced with Venus-NS of PR8,and the virus was adapted to mice as described for MA−Venus−PR8. A stockof MA−Venus−HPAI virus was made in MDCK cells. The set of theseinfluenza viruses carrying various fluorescent proteins was collectivelytermed “Color-flu”.

Mouse Experiments

Female, 6-week-old C57BLU6 (‘B6’) mice were purchased from Japan SLC,Inc. (Shizuoka, Japan). Mice were intranasally inoculated with Color-fluviruses, at the dosages indicated in the figure panels, in 50 μL of PBSunder sevoflurane anesthesia, and body weights and survival weremonitored for 14 days. Lungs were harvested from PBS-inoculated orColor-flu-infected mice for virus titration, flow cytometric analysis,and histological experiments at the times indicated in the figurepanels. All animal experiments were performed in accordance with theregulations of the University of Tokyo Committee for Animal Care and Useand were approved by the Animal Experiment Committee of the Institute ofMedical Science of the University of Tokyo.

Histology and Cytology

Lungs were fixed in 4% paraformaldehyde (PFA) phosphate buffer solution.Fixed tissues were embedded in OCT compound (Sakura Finetek, Tokyo,Japan), frozen by liquid N2 and stored at −80° C. Cryostat 6-μm sectionswere treated for 30 minutes with PBS containing 1% BSA (PBS-BSA) toblock nonspecific binding, and then incubated with phycoerythrin(PE)-Mac3 (M3/84, BD Biosciences. San Jose, Calif.). To examine thecytology of the MDCK cells, cells were infected with Color-flu virus andthen fixed in 4% PFA phosphate buffer solution. Nuclei were stained withHoechst33342 (Invitrogen, Carlsbad, Calif.). Sections and cells werevisualized by using a confocal microscope (Nikon A1, Nikon, Tokyo,Japan), controlled by NIS-Elements software. For quantitativemulti-color imaging analysis, the slides were visualized by use of aninverted fluorescence microscope (Nikon Eclipse TS100) with a Nuance FXmultispectral imaging system with InForm software (PerkinElmer, Waltham,Mass.).

Whole-Mount Imaging of Lung Tissue

Mice were euthanized and intracardially perfused with PBS to removeblood cells from the lung. The lungs were isolated after intratrachealperfusion with 4% PFA phosphate buffer solution. The lung tissues werecleared with SCALEVIEW-A2 solution (Olympus, Tokyo, Japan) according tothe manufacturer's instructions. Images were acquired by using a stereofluorescence microscope (M205FA, Leica Microsystems, Wetzlar, Germany)equipped with a digital camera (DFC365FX, Leica Microsystems).

Two-Photon Laser Microscopy

A total of 10⁵ PFU of MA−eGFP−PR8 was intranasally inoculated into B6mice. To label lung macrophages, 50 μL of PE−CD11b (M1/70, BioLegend,San Diego, Calif.) was injected intravenously to the mice at day 3 p.i.Thirty minutes after the antibody injection, the lungs of the mice wereharvested. The kinetics of eGFP- and PE-positive cells in the lungs wereimaged with a multi-photon microscope (LSM 710 NLO, Carl Zeiss,Oberkochen, Germany). During the analysis, the lungs were maintained incomplete medium (RPMI 1640 with 10% fetal calf serum) in a humid chamber(37° C., 5% CO₂). The data were processed with LSM software Zen 2009(Carl Zeiss). For three-dimensional imaging of HPAI virus-infected lungtissues, B6 mice were intranasally inoculated with 10⁵ PFU ofMA−Venus−HPAI virus. The lung tissues were collected from the mice atday 2 p.i., and treated with SCALEVIEW-A2 solution (Olympus) to maketissues transparent as described above. Three-dimensional images of lungtissues were obtained from a multi-photon microscope (Nikon A1R MP).

Flow Cytometric Analysis and Cell Sorting

To obtain single-cell suspensions, lungs were dissociated withCollagenase D (Roche Diagnostics, Mannheim, Germany; finalconcentration: 2 g/mL) and DNase I (Worthington Biochemical, Lakewood,N.J.; final concentration: 40 U/mL) for 30 minutes at 37° C. by grindingthe tissue through nylon filters (BD Biosciences). Red blood cells(RBCs) were lysed by treatment with RBC lysing buffer (Sigma Aldrich,St. Louis, Mo.). To block nonspecific binding of antibodies, cells wereincubated with purified anti-mouse CD16/32 (Fc Block, BD Biosciences,San Diego, Calif.). Cells were stained with appropriate combinations offluorescent antibodies to analyze the population of each immune cellsubset. The following antibodies were used: anti-CD45 (30-F11:eBioscience, San Diego, Calif.), anti-CD11b (M1/70: BioLegend),anti-F4180 (BM8: eBioscience), and anti-CD11c (HL3: BD Biosciences). Allsamples were also incubated with 7-aminoactinomycin D (Via-Probe, BDBiosciences) for dead cell exclusion. Data from labeled cells wereacquired on a FACSAria II (BD Biosciences) and analyzed with FlowJosoftware version 9.3.1 (Tree Star, San Carlos, Calif.). To isolateVenus-positive and -negative macrophages from lungs, stained cells weresorted using a FACSAria II (BD Biosciences).

Microarray Analysis

Total RNA of sorted macrophages was extracted using TRIzol reagent (LifeTechnologies, Carlsbad, Calif.) and precipitated with isopropanol. RNAamplification was performed using the Arcturus Riboamp Plus RNAAmplification Kit (Life technologies) in accordance with themanufacturer's instructions. RNA was labeled by using the Agilent LowInput Quick Amp Labeling kit, one color (Agilent Technologies, SantaClara, Calif.) and hybridized to the SurePrint G3 Mouse GE 8X60Kmicroarray (Agilent Technologies). Arrays were scanned with a DNAMicroarray Scanner with SureScan High-Resolution Technology, (G2565CA;Agilent Technologies), and data were acquired using Agilent FeatureExtraction software ver. 10.7.3.1. (Agilent Technologies). Probeannotations were provided by Agilent Technologies (AMADID 028005). Probeintensities were background corrected and normalized using thenormal-exponential and quantile methods, respectively. The log₂ of theintensities were then fit to a linear model that compared the groups ofinterest³⁴. All reported p values were adjusted for multiple hypothesiscomparisons using the Benjamini-Hochberg method. Transcripts wereconsidered differentially expressed if there was at least a 2-foldchange in the mean probe intensity between contrasts with an adjustedp<0.01. Hierarchical clustering was performed in R. The resultant geneclusters were then analyzed with ToppCluster (Kaimal et al., 2010) toidentify gene annotations that were enriched in each cluster. Thereported scores are the −log₁₀ of the Benjamini-Hochberg adjustedp-value.

Western Blot Analysis

Whole lysates of MDCK cells were electrophoresed throughSDS-polyacrylamide gels (Bio-Rad Laboratories, Hercules, Calif.) andtransferred to a PVDF membrane (Millipore, Billerica, Mass.). Themembrane was blocked with Blocking One (Nacalai Tesque, Kyoto, Japan)and incubated with a rabbit anti-GFP polyclonal antibody (MBL, Nagoya,Japan), mouse anti-NS1 antibody (188/5), rabbit antiserum toA/WSN/33(H1N1)(R309) or mouse anti-actin antibody (A2228;Sigma-Aldrich), followed by HR-conjugated anti-mouse or anti-rabbit IgGantibody (GE Healthcare, Waukesha, Wis.). After the membrane was washedwith PBS-Tween, specific proteins were detected using ECL Plus WesternBlotting Detection System (GE Healthcare. The specific protein bandswere visualized by the use of the VersaDoc Imaging System (Bio-Rad).

Results

To generate a fluorescent influenza virus expressing a reporter proteinfused to the NS1 open reading frame, Venus was chosen, a GFP variantwith eight mutations including F46L, which improves chromophoreformation and increases brightness compared with GFP (Wagai et al.,2002). As expected based on previous findings of attenuation forinfluenza viruses expressing reporter proteins (Kittel et al., 2004;Shinhya et al., 2004), the mouse pathogenicity of A/Puerto Rico/8/34(PR8; H1N1) virus expressing Venus (WT−Venus−PR8) was substantiallylower than that of wild-type PR8 (WT−PR8); the dose required to kill 50%of infected mice (MLD₅₀) was more than 10^(4.5) plaque-forming units(PFU) for WT−Venus−PR8 compared with 10^(2.5) PFU for WT-PR8.WT−Venus−PR8 was serially passaged in C57BL/6 (B6) mice. After sixconsecutive passages, a variant (designated MA−Venus−PR8; possessing aT-to-A mutation at position 380 of the hemagglutinin protein, and anE-to-D mutation at position 712 of the polymerase subunit PB2) wasidentified with appreciably higher pathogenicity (MLD₅₀=10^(3.5) PFU)compared with WT−Venus−PR8, although it was still less pathogenic thanthe original PR8 virus (FIG. 1A). To assess the replicative ability ofMA−Venus−PR8 in mouse lungs, B6 mice were intranasally infected with 10⁴PFU of MA−Venus−PR8 or PR8 virus. At all time points tested, the lungvirus titers were similar for MA−Venus−PR8- and PR8-infected mice (FIG.1B). To test the stability of Venus expression, plaque assays wereperformed using lung homogenate from infected mice and found that onlyone of 150 plaques on each of days 3, 5, and 7 post-infection (p.i.) wasVenus-negative, attesting to the high genetic stability of Venusexpression in this recombinant virus. In contrast, only 70% of NS1-GFPvirus expressed the reporter protein (Manicassamy et al., 2010). Therobust virulence and genetic stability of MA−Venus−PR8 indicate thatthis virus represents a highly attractive reporter system to visualizeinfluenza virus-infected cells in vivo.

TABLE 1 Replication and virulence of Color-flu in mice*. Mean virustiter (log10 PFU/g ± s.d.) in the mouse lung on the indicated day p.i.Virus Day 3 p.i. Day 5 p.i. Day 7 p.i. MLD₅₀ (PFU) MA-eCFP-PR8  8.1 ±0.2** 8.0 ± 0.1 6.3 ± 0.1 10^(3.0) MA-eGFP-PR8 8.6 ± 0.1 8.3 ± 0.1 6.3 ±0.1 10^(3.5) MA-Venus-PR8 8.6 ± 0.2 8.4 ± 0.1 6.5 ± 0.3 10^(3.3)MA-mCherry-PR8  7.7 ± 0.3** 7.5 ± 0.7 6.1 ± 0.4 10^(2.7) WT-Venus-PR8 5.6 ± 0.3**  5.3 ± 0.3**  5.2 ± 0.2** >10^(4.3)  WT-PR8 8.8 ± 0.1 8.2 ±0.5 6.9 ± 0.2 10^(2.5) MA-PR8 8.9 ± 0.1 9.0 ± 0.0  7.9 ± 0.2** 10^(2.3)*B6 mice were inoculated intranasally with 10⁴ PFU of each virus in a 50mL volume. Three mice from each group were killed on days 3, 5 and 7p.i., and virus titres in the lungs were determined in MDCK cells.**Statistical significance was calculated by using the Student's t-test;the P value was <0.01 compared with the titres in the lungs of miceinfected with WT-PR8 virus.

To increase the versatility of fluorescent influenza viruses as imagingtools, additional MA−PR8 variants were generated that expresseddifferent spectral GFP mutants, namely, eCFP (ex. 434 nm, em. 477 nm)and eGFP (ex. 489 nm, em. 508 nm) (Patterson, 2001). A mCherry variant(ex. 587 nm, em. 610 nm), which emits fluorescence at a longerwavelength than Venus (ex. 515 nm, em. 528 nm) (Nagai et al., 2002;Shaner et al., 2004), was also generated. These influenza virusesencoding the multi-spectral fluorescent reporter proteins werecollectively named “Color-flu”. To determine the pathogenicity ofColor-flu viruses, the virus titres in mouse lung tissues and the MLD₅₀values of MA−eCFP, eGFP and mCherry-PR8 were compared with those ofMA−Venus−PR8 and MA−PR8. All of virus strains showed comparatively highreplication in the lungs and the MLD₅₀ values were similar among theColor-flu viruses (Table 1). The stability of the fluorescent expressionof the Color-flu viruses was tested in vivo and in vitro by plaqueassay. When virus was collected from the lungs of mice on day 7 p.i.,the percentages of fluorescent-positive plaques were 98.0%(MA−eCFP-PR8), 100.0% (MA−eGFP−PR8) and 96.4% (MA−mCherry−PR8). Thepercentages of fluorescent-positive plaques in the sample from theculture medium of MDCK cells after 72 hours p.i. was found to be 100.0%(MA−eCFP-PR8), 99.2% (MA−eGFP−PR8) and 98.2% (MA−mCherry−PR8). Inaddition, the stability of an NS1-fluorescent protein chimera invirus-infected cells was examined by infecting MDCK cells withMA−Venus−PR8 virus and detecting NS1−Venus chimeric protein by usinganti-GFP and anti-NS1 antibodies. The NS1−Venus chimeric protein was notdegraded until the time point examined (that is, 12 hours p.i.),indicating that the fluorescent signal is mainly emitted from theNS1-fluorescent protein chimera and not from degradation products incells infected with Color-flu viruses. These findings indicate that thepathogenicity and stability of the Color-flu viruses were not affectedby the different fluorescent reporter genes.

To assess the expression of Color-flu viruses in mouse lungs, wecollected lungs from B6 mice infected with each of the Color-flu virusesand processed them for visualization as described in the Methodssection. All four colors were clearly visible in whole transparent lungtissue when analyzed with a fluorescent stereomicroscope (FIG. 2A).Fluorescent signals were mainly seen in the bronchial epithelial layerat day 3 p.i. At day 5 p.i., fluorescent signals extended to theperipheral alveolar regions. These data indicated that all fourColor-flu viruses are useful for analyzing the distribution of influenzavirus-infected cells in mouse lungs. To assess the expression ofColor-flu viruses in mouse lungs, lungs were collected from B6 miceinfected with each of the Color-flu viruses and processed them forvisualization as described in the Methods section. All four colors wereclearly visible in whole transparent lung tissue when analyzed with afluorescent stereomicroscope (FIG. 2A). Fluorescent signals were mainlyseen in the bronchial epithelial layer at day 3 p.i. At day 5 p.i.,fluorescent signals extended to the peripheral alveolar regions. Thesedata indicate that all four Color-flu viruses are useful for analyzingthe distribution of influenza virus-infected cells in mouse lungs.

Next, the Nuance™ spectral imaging system was employed to test whetherthe fluorescent signals of all four Color-flu viruses could be detectedsimultaneously. Lung tissues were collected from B6 mice intranasallyinoculated with a mixture of the four strains (2.5×10⁴ PFU each in atotal volume of 50 μL). Analysis of lung sections obtained at days 2 and5 p.i. showed that the fluorescent signals of all four Color-flu viruseswere distinguishable from each other (FIG. 2B). At day 2 p.i., clustersof the same fluorescent color were found in bronchial epithelial cells,suggesting local spread of the individual viruses. At this time point, alimited number of alveolar cells were infected. At day 5 p.i., wedetected a cluster of alveolar cells expressing a single fluorescentprotein, indicative of the initiation of infection with a single virusand its local spread (FIG. 2B). Interestingly, epithelial cellssimultaneously expressing two or three fluorescent proteins weredetected, albeit at a low frequency, suggesting co-infection of thesecells (FIG. 2C). The ability to visualize cells co-infected withdifferent influenza viruses in vivo is a major advance in technology andwill allow insights into influenza co-infection and reassortmentprocesses.

Next, the utility of Color-flu viruses was tested for the analysis ofhost responses to infection. Since macrophages are involved in innateimmunity and acute inflammation in influenza virus-infected lungs, lungsections that were stained with an antibody to macrophages (PE-Mac3)were examined by using confocal microscopy. Macrophages infiltratedregions containing Venus-positive bronchial epithelial cells at day 2p.i. of mice with MA−Venus−PR8 (FIG. 3A); by contrast, only a fewMac3-positive cells were detected in the alveoli of lungs frommock-infected animals. On the basis of this finding, live imaging wasemployed to further study the interaction between influenzavirus-infected epithelial cells and macrophages in mouse lungs. In thelung tissue of naive B6 mice, CD11b+ alveolar macrophages were detectedby use of a two-photon laser microscope. Most of these macrophages didnot migrate (i.e., showed little movement) during the observation period(49 minutes; data not shown). In mice infected with MA−eGFP−PR8 virus,many CD11b+ macrophages appeared to be ‘attached’ to eGFP-positiveepithelial cells (data not shown); moreover, some of these eGFP-positiveepithelial cells exhibited blebbing similar to apoptotic cells.Interestingly, a number of CD11b+ macrophages quickly moved around theeGFP-positive epithelial cells, suggesting possible macrophage responsesto inflammatory signals such as IFNs or chemokines. The present systemcan thus be used to monitor the in vivo interactions betweenvirus-infected and immune cells.

A number of studies have assessed the transcriptomics and proteomicsprofiles of influenza virus-infected mice (Go et al., 2012; Zhao et al.,2012). Since these studies used whole lung samples, the results are thesum of virus-infected and uninfected cells, leading to the dilution ofhost responses and not allowing one to distinguish the profiles ofinfected cells from those of uninfected, bystander cells. As a firststep to overcome this shortcoming, macrophages (known to be infected byinfluenza viruses (FIG. 3B)) from the lungs of mice infected withMA−Venus−PR8 were sorted on the basis of their fluorescent proteinexpression and performed microarray analysis. Macrophages isolated fromthe lungs of mice inoculated with PBS (naive macrophages) served ascontrols. In fluorescent-positive macrophages, 6,199 transcripts weredifferently expressed relative to naive macrophages. By contrast, influorescent-negative macrophages obtained from infected mice, only 4,252transcripts were differentially expressed relative to the naivemacrophages. This difference likely reflects differences in genetranscription induced by active influenza virus infection. However, itshould be noted that the fluorescent-negative cell populations obtainedfrom infected animals may have included infected cells in which thefluorescent signal had not yet been detected as would be expected at anearly stage of virus infection. In fact, confocal microscopy revealedthat it took 9 hours to detect fluorescent protein expression in themajority of MDCK cells. Hierarchical clustering of differentiallyexpressed transcripts, followed by functional enrichment analysis ofeach cluster, indicated that both fluorescent-positive andfluorescent-negative macrophages obtained from infected animals exhibitactivation of pathways associated with the immune response, cytokineproduction, and inflammation (FIG. 3D, green cluster). The upregulationof these pathways in the fluorescent-negative cells may have resultedfrom cell activation by IFN and cytokines released from infected cells,and/or from cells that were at an early stage of virus infection (asdiscussed earlier). Yet, a subset of enriched annotations, for example,type I IFN-mediated signaling (FIG. 3D, light blue cluster), includedtranscripts that were more highly expressed in fluorescent-positivemacrophages. In addition, it was observed that type I IFN genes wereamong the most upregulated transcripts in the fluorescent-positivemacrophages (FIG. 3E). Taken together, this enhanced type I IFN activityis consistent with the suggestion that the fluorescent-positive cellshad been infected whereas the fluorescent-negative cells included bothuninfected (but potentially ‘stimulated’) cells and cells at earlystages of influenza virus infection. Indeed, it took at least 5 hours todetect fluorescent protein expression after infection with Color-fluviruses, although all of the fluorescent proteins (that is, eCFP, eGFP,Venus, and mCherry) were detectable in the majority of cells by 9 hoursp.i. These findings open new avenues in infectious disease research tocompare gene expression (or other types of expression) patterns ofreporter protein-positive cells with those of reporter protein-negativecells (but potentially stimulated by released cytokines and/or are at anearly stage of infection).

Finally, as discussed in more detail in Example II, it was testedwhether the concept of mouse-adapted fluorescent influenza viruses couldbe applied to other influenza virus strains, such as highly pathogenicavian influenza A (H5N1) (HPAI) viruses, which are a research prioritydue to the threat they pose to humans. An MA−Venus−HPAI virus based onA/Vietnam/1203/2004 (VN1203; H5N1) was generated, employing the samestrategy used to create MA−Venus−PR8; however, the PR8 NS gene was usedto express NS1−Venus chimeric protein because Venus virus with theVN1203 NS gene did not contribute to pathogenicity in mice. Thepathogenicity of MA−Venus−HPAI virus for B6 mice was comparable to thatof VN1203, with MLD₅₀ values for both viruses being less than 5 PFU(FIG. 4A and Hatta et al., 2007). MA−Venus−HPAI virus also shared withother HPAI viruses the ability to spread systemically and replicate invarious organs including spleen, kidney, and brain (FIG. 4B and Hatta etal., 2007). Moreover, taking advantage of the strong fluorescent signalemitted by MA−Venus−HPAI virus-infected cells, a three-dimensional imageof an HPAI virus-infected bronchus deep inside the lung tissues wassuccessfully constructed (FIG. 4C and data not shown). This type ofthree-dimensional imaging analysis will improve the understanding of thespatial distribution of influenza virus-infected bronchi. When thedistribution of virus-infected cells was compared between HPAI virus andPR8-infected lungs, it was found that HPAI virus spreads from thebronchial epithelium to alveolar sites more quickly than did PR8 (FIGS.4C and D). By using flow cytometric analysis, it was found thatCD45-negative, non-hematopoietic cells and F4/80-positive macrophagesmore frequently expressed Venus in the lungs of mice infected withMA−Venus−HPAI virus than in the lungs of animals inoculated withMA−Venus−PR8 (FIGS. 4E and F), supporting findings that H5N1 HPAIviruses induce more severe inflammatory responses in the lung than doesPR8, demonstrating the utility of Color-flu viruses for comparativestudies of influenza pathogenesis.

Discussion

In this study, Color-flu viruses were generated to study influenza virusinfections at the cellular level. Color-flu viruses combine severalimprovements over existing systems, including robust viral replication,virulence, stable fluorescent protein expression, and a set of fourdifferent colors that can be visualized simultaneously. Color-fluviruses are applicable to all influenza virus strains. Theseimprovements allowed global transcriptomics analyses of infected andbystander cells and, for the first time, live-imaging of influenzavirus-infected cells in the mouse lung.

Previous versions of fluorescent influenza viruses (Kittel et al., 2004;Shinya et al., 2004) including our original construct (i.e.,WT−Venus−PR8) were appreciably attenuated in mice. These attenuatedfluorescent viruses may still be useful for identifying initial targetcells. However, the immune responses elicited by these highlyattenuated, non-lethal viruses most likely differ considerably fromthose of the mouse-lethal parent virus, making their use forpathogenesis studies problematic. This problem was solved by passagingviruses in mice. This strategy proved to be successful for two differentinfluenza virus strains, suggesting its broad applicability. A seconddrawback of previously tested fluorescent influenza viruses is thegenetic instability of the added reporter protein (Manicassamy et al.,2010). However, almost 100% of virus plaques examined from mouse lungsamples on day 7 post-infection expressed the reporter protein.

At present, Color-flu viruses cannot be monitored in live animalsnon-invasively because fluorescent reporter proteins must be within a“biological optical window (650-900 nm)” to be detected for imaging oftissues in live animals using fluorescent probes (Weisslander, 2001;Jobsis, 1977), and none of the fluorescent reporter proteins includingmCherry, which has the longest emission among the reporter proteins ofColor-flu, is inside this biological optical window. Heaton et al.(2013) generated a luciferase reporter-expressing influenza virus thatcan be used to monitor virus replication in live animals; however, thissystem needs systemic inoculation of substrate into the animals at everyobservation point. In addition, the resolution of their imaging system(based on the IVIS® system) is not adequate for the analysis of cellularimmune mechanisms in vivo, which we are able to achieve with the presentsystem.

Newer technologies for imaging analysis (Ghoznari et al., 2013) haveenabled the development of a set of four different influenza colorvariants that can be distinguished from one another by using Nuance™,hence allowing their simultaneous detection. In fact, our pilot studyidentified lung epithelial cells expressing two or three differentfluorescent proteins (FIG. 2C). This may be the first visualization ofmouse lung cells infected with more than one influenza virus strain. Infuture studies, these color variants could be used to addresslong-standing questions in influenza virus research, such as thefrequency of viral co-infections in vivo, which may be critical tobetter understand influenza virus reassortment and, hence, thegeneration of novel influenza viruses such as the pandemic viruses of1957 (Schaltissek et al., 1978; Kanaoka et al., 1989), 1968 (Schaltisseket al., 1978; Kanaoka et al., 1989), and 2009 (Smith et al., 2009; Itohet al., 2009).

By employing the described tool sets, influenza virus-infected cellswere detected in whole lung tissues of mice, allowing the observation ofthe location and distribution of influenza viruses in the lung.Moreover, interactions of virus-infected epithelial cells with immunecells were observed. Such studies will allow direct monitoring influenzadisease progression from acute bronchitis to severe viral pneumonia,which causes considerable morbidity and mortality in highly pathogenicinfluenza virus infections (Gambotto et al., 2008; Shieh et al., 2009).

In conclusion, Color-flu viruses in combination with advanced imagingtechnologies allow for detection at the cellular level in animals.

Example II

As disclosed in Example I, an H5N1 virus with the Venus (Nagai et al.,2002) (a variant of eGFP) reporter gene (designated wild-type Venus−H5N1virus, and abbreviated as WT−Venus−H5N1 virus) was prepared usingreverse genetics; this virus showed moderate virulence and low Venusexpression in mice. After six passages in mice a mouse-adaptedVenus−H5N1 virus was acquired (abbreviated as MA−Venus−H5N1 virus) thatstably expressed high levels of Venus in vivo and was lethal to mice; adose required to kill 50% of infected mice (MLD₅₀) was 3.2plaque-forming units (PFU), while that of its parent WT−Venus−H5N1 viruswas 103 PFU. However, the mechanism for this difference in virulence andVenus stability was unclear.

In this study, the molecular mechanism that determines the virulence andVenus stability of Venus−H5N1 virus in mice was explored. By usingreverse genetics, various reassortants between WT−Venus−H5N1 andMA−Venus−H5N1 virus were rescued and their virulence in mice examined toidentify determinants for pathogenicity. Further, the determinants forVenus expression and Venus stability in vitro and in vivo wereinvestigated. The findings further the understanding of thepathogenicity of influenza virus in mammals and will benefit thedevelopment of influenza virus-related vaccines and therapy.

Materials and Methods

Cells. Human embryonic kidney 293 and 293T cells were maintained inDulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum, andMadin-Darby canine kidney (MDCK) cells were maintained in minimalessential medium (MEM) supplemented with 5% newborn calf serum. All thecells were incubated at 37° C. in 5% CO₂.

Construction of Plasmids. Plasmids for virus rescue was constructed asdescribed in Neumann et al. (1999). To measure viral polymeraseactivity, the open reading frames of the PB1, PB2, PA, and NP ofinfluenza virus were amplified by PCR with gene-specific primers andcloned into the pCAGGS/MCS protein expression plasmid (Dias et al.,2009). The primer sequences are listed below:

TABLE 2  Gene Primer sequence (5′---3′) VN1203- UpperCCCATCGATACCATGGAGAGGATAAAAGAATTACG P82 AGATC (SEQ ID NO: 20) LowCTAGCTAGCCTACTAATTGATGGCCATCCGAATTC TTTTG (SEQ ID NO: 21) VN1203- UpperTACGAGCTCACCATGGATGTCAATCCGACTTTACT PK TTT (SEQ ID NO: 22) LowCTAGCTAGCCTACTATTTTTGCCGTCTGAGTTCTT CAATG (SEQ ID NO: 23) VN1203- UpperCCCATCGATACCATGGAAGACTTTGTGCGACAATG PA C (SEQ ID NO: 24) LowCTAGCTAGCCTACTATTTCAGTGCATGTGCGAGGA AGGA (SEQ ID NO: 25) VN1203- UpperTTCATCGATACCATGGCGTCTCAAGGCACCAAAC  NP (SEQ ID NO: 26) LowCGCGCTAGCCTATTAATTGTCATACTCCTCTGCAT TGTCT (SEQ ID NO: 27)All of the constructs were completely sequenced to ensure the absence ofunwanted mutations.

Plasmid-Based Reverse Genetics. Influenza A viruses were generated byusing plasmid-based reverse genetics, as described previously (Murakami,2008; Ozawa et al.; 2007). Viral titers of the rescued viruses weredetermined by use of plaque assays in MDCK cells. All rescued viruseswere sequenced to confirm the absence of unwanted mutations.

Mouse Experiments. Six-week-old female C57/BL6 (B6) mice (Japan SLC,Inc., Shizuoka, Japan) were used in this study. To measure viralreplication in mice, six mice in each group were anesthetized withisoflurane and then intranasally inoculated with 105 PFU (50 μL) ofvirus. On days 1 and 3 post-infection (p.i.) three mice were euthanized,and their organs including the lungs, kidneys, spleens, and brains werecollected and titrated in MDCK cells. To determine the 50% mouse lethaldose (MLD₅₀) of the viruses, four mice from each group were inoculatedintranasally with 10-fold serial dilutions containing 10⁰ to 10⁵ PFU (50L) of virus, respectively. Body weight and survival were monitored dailyfor 14 days. The MLD₅₀ was calculated by using the method of Reed andMuench (1938). All mouse experiments were performed in accordance withthe University of Tokyo's Regulations for Animal Care and Use and wereapproved by the Animal Experiment Committee of the Institute of MedicalScience, the University of Tokyo.

Virus Passage in Mice and MDCK Cells. Mouse adaptation of virus wasperformed as described in Example I. For virus passages in MDCK cells,confluent MDCK cells were infected with virus at a multiplicity ofinfection (MOI) of 0.0001. At 48 hours post-infection (hpi) thesupernatants were collected and titered in MDCK cells. The new,harvested viruses were used to infect MDCK cells for the next passage.This procedure was repeated five times.

Growth Kinetics Assays. Each virus was inoculated into triplicate wellsof subconfluent MDCK cells at an MOI of 0.0001. The cells weresupplemented with MEM containing 0.3% bovine serum albumin (BSA) and 1μg/mL tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK) trypsin andincubated at 37° C. in 5% CO₂. Culture supernatants were harvested atthe indicated hours post-infection. The viral titers of the supernatantsat the different time-points were determined by use of plaque assays inMDCK cells.

Mini-Genome Luciferase Assay. Polymerase activity was tested with amini-genome assay by using the dual-luciferase system as previouslydescribed in Murakami (2008) and Ozawa et al. (2007). Briefly, 293 cellswere transfected with viral protein expression plasmids for NP, PB1,PB2, and PA from the WT−Venus−H5N1 or MA−Venus−H5N1 virus (0.2 μg each),with a plasmid expressing a reporter vRNA encoding the fireflyluciferase gene under the control of the human RNA polymerase I promoter(pPoll/NP(0)Fluc(0), 0.2 μg), and with pRL-null (Promega, 0.2 μg), whichencodes the Renilla luciferase, as an internal transfection control. At24 hours post-transfection, cell lysate was prepared with theDual-Luciferase Reporter Assay System (Promega) and luciferase activitywas measured by using the GloMax 96 microplate luminometer (Promega).The assay was standardized against Renilla luciferase activity. Allexperiments were performed in triplicate.

Laboratory facility. All studies with H5N1 viruses were performed inenhanced biosafety level 3 containment laboratories at the University ofTokyo (Tokyo, Japan), which are approved for such use by the Ministry ofAgriculture, Forestry, and Fisheries, Japan.

Statistical analysis. The data were analyzed by using the R software(www.r-project.org), version 3.1. For comparisons of measurements frommultiple groups collected at a single time point, we used one-way ANOVAfollowed by Tukey's Post hoc test. For comparisons of multiple groupswith measurements collected independently at different time-points(i.e., viral growth curves from mice, collected in MDCK cells), we usedtwo-way ANOVA followed by Tukey's Post hoc test. For comparisons ofmultiple groups with dependent measurements (i.e., viral growth 7 curvesin cell culture for which aliquots were collected from the same cultureat different time points), a linear mixed-effects model was fitted tothe data using the R package NLME, and the time, the virus strain, andthe interaction between these two factors were considered. Next, acontrast matrix was built to compare the strains in a pairwise fashionat the same time points (e.g., group_1 vs group_2 at 24 hourspost-infection, group_1 vs group_3 at 24 hours post-infection, group_2vs group_3 at 24 hours post-infection), using the R package PHIA.Because the comparisons were performed individually, the final p-valueswere adjusted by using Holm's method to account for multiplecomparisons. In all cases, the results were considered statisticallysignificant if we obtained In all cases, the results were consideredstatistically significant if we obtained p-values (or adjustedp-values)<0.05.

Sequence analysis. The PB2 and PA sequences from the NCBI InfluenzaVirus Database were aligned by using the MUSCLE program (Edgar, 2004),with the default parameters and a maximum of 100 iterations. Thealignment was visualized by using Clustal X (Larkin et al., 2007), andthe frequency of amino acid occurrences at specific positions wasdetermined by using custom written Per scripts.

Results Comparison Between WT−Venus−H5N1 and MA−Venus−H5N1 Viruses.

As in Example 1, the NS segment of A/Viet Nam/1203/2004 (H5N1)(abbreviated as VN1203) was substituted with a Venus-fused NS segment ofVenus−PR8 virus by using reverse genetics, and acquired an H5N1 virusthat expressed the Venus fluorescent reporter gene (WT−Venus-1H51N11virus). A pathogenicity analysis in mice revealed that this virusexhibited attenuated virulence in mice compared with that of theparental VN1203 with an MLD₅₀ value of 10³ PFU (compared with 0.7 PFUfor VN1203) (FIGS. 6A and 6B and Hatta et al. (2007)). Moreover,WT−Venus−H5N1 virus mainly replicated in respiratory organs (Table 3),and its Venus expression was very weak in both MDCK cells (FIG. 7) andin mice after virus infection (FIG. 8).

TABLE 3 Replication and virulence of H5N1 reassortants and mutants inmice^(a) Mean virus titer (log₁₀ PFU/g ± SD) on the indicated day p.i.Lung Spleen Kidney Brain Day 1 Day 3 Day 1 Day 3 Day 1 Day 3 Day 1 Day 3Virus p.i. p.i. p.i. p.i. p.i. p.i. p.i. p.i. WT-Venus-H5N1 6.5 ± 0.2  6.3 ± 0.2     — ^(b) — — — — — MA-Venus-H5N1 9.1 ± 0.1 ^(c) 8.9 ± 0.0^(c) 3.4 ± 0.2 6.5 ± 0.1 2.4 ± 0.1 4.1 ± 0.1 — 2.8 ± 0.6 RG-MA 9.1 ± 0.1^(c) 9.2 ± 0.1 ^(c) 2.8 ± 0.4 7.1 ± 0.0 2.3, —, — 4.0 ± 0.1 — 2.3 ± 0.2WT + MA-PB2 7.7 ± 0.0 ^(c) 8.0 ± 0.1 ^(c) — 4.1, 4.4, — — — — — WT +MA-PA 7.1 ± 0.1 ^(c) 6.8 ± 0.1 ^(d) — — — — — — WT + MA- 8.6 ± 0.3 ^(c)8.7 ± 0.1 ^(c) 2.9 ± 0.4 6.8 ± 0.0 2.0, 1.7, — 4.3 ± 0.7 — 2.7 ± 0.8(PB2 + PA) ^(a)Six-week-old SPF C57/BL6 mice were inoculatedintranasally with 10⁵ PFU of each virus in a 50-μl volume. Three micefrom each group were euthanized on days 1 and 3 p.i., and virus titerswere determined in samples of lung, spleen, kidney, and brain in MDCKcells. ^(b) —, no virus was isolated from the sample. ^(c) P value was<0.01 compared with the titers in the lungs of mice infected withWT-Venus-H5N1 virus. ^(d) P value was <0.05 compared with the titers inthe lungs of mice infected with WT-Venus-H5N1 virus.

After six passages of WT−Venus−H5N1 virus in mice, MA−Venus−H5N1 viruswas obtained. MA−Venus−H5N1 virus was lethal to mice with an MLD₅₀ of3.2 PFU (Example 1). This virus replicated systemically in mice; on day1 p.i., high viral titers were detected in lungs, spleens, and kidneys,and on day 3 p.i. virus could be detected in brains (Table 3). Moreoverwe detected high Venus expression of MA−Venus−H5N1 virus in MDCK cells(FIG. 7) and in mice (FIG. 8). Therefore, compared with WT−Venus−H5N1,MA−Venus−H5N1 virus showed much higher pathogenicity in mice and ahigher replicative ability. Moreover, this virus exhibited high Venusexpression during its replication in vitro and in vivo.

To identify the genetic mutations that had occurred during mouseadaptation, the genome of MA−Venus−H5N1 virus was sequenced and comparedto that of WT−Venus−H5N1 virus. At the amino acid level, a total ofseven differences were found between the two viruses in their PB1, PB2,PA, NA, M2, and NS1 genes (Table 4). Thus single or multiple amino-acidchanges among these seven different amino acids may contribute to thedifference in virulence in mice and in Venus expression between thesetwo viruses.

TABLE 4 Amino acid differences between WT- Venus-H5N1 and MA-Venus-H5N1viruses. Viral Amino acid Amino acid in: segment position WT-Venus-H5N1MA-Venus-H5N1 PB2 25 Val (V) Ala (A) PB1 737 Lys (K) Arg (R) PA 443 Arg(R) Lys (K) NA 35 Ser (S) Arg (R) 284 Val (V) Leu (L) M2 64 Ala (A) Asp(D) NS1 167 Pro (P) Ser (S)

V25A of PB2 and R443K of PA Determine the Pathogenicity and VenusExpression of Venus−H5N1 Virus in Mice.

To investigate the genetic basis for the difference in the virulence andVenus expression of Venus−H5N1 virus after mouse adaptation, a reversegenetics system was established for MA−Venus−H5N1 virus, which was namedRG−MA virus. RG−MA virus exhibited similar viral titers in organs, MLD₅₀value (FIGS. 6C and 6D and FIG. 9), and Venus expression in mice (FIG.9) to those of its original virus (MA−Venus−H5N1 virus).

To identify the amino acids responsible for the difference in virulenceand Venus expression between WT−Venus−H5N1 and MA−Venus−H5N1 virus, sixsingle-gene recombinant viruses were generated, each bearing the PB2,PB1, PA, NA, M, or NS gene from MA−Venus−H5N1 virus and the other sevengenes from WT−Venus−H5N1 virus. The recombinant viruses that containedthe PB1, NA, or NS gene of MA−Venus−H5N1 virus (designated WT+MA−PB1,WT+MA−NA, or WT+MA−NS) displayed similar pathogenicity in mice to thatof WT−Venus−H5N1 (MLD₅₀, 103 PFU) (FIG. 9), whereas the reassortantswith the PB2, PA, or M gene of MA−Venus−H5N1 (designated WT+MA−PB2,WT+MA−PA, or WT+MA−M) exhibited higher pathogenicity in mice thanWT−Venus−H5N1 (FIG. 9). WT+MA−PB2 and WT+MA−PA replicated moreefficiently in mouse lungs than did WT−Venus−H5N1; moreover virus wasdetected in the spleens of two of three mice infected with WT+MA−PB2.

The effect of the PB2, PA, or M genes derived from WT−Venus−H5N1 on thevirulence of MA−Venus−H5N1 was also examined by generating threesingle-gene recombinant viruses, each containing the PB2, PA, or M genefrom WT−Venus−H5N1 virus and the remaining segments from MA−Venus−H5N1virus (designated MA+WT−PB2, MA+WT−PA, or MA+WT−M). The MLD₅₀ values ofMA+WT−PB2 and MA+WT−PA were 10²³ PFU, significantly higher than that ofMA−Venus−H5N1 (MLD₅₀, 3.2 PFU), whereas the virulence of MA+WT−M in micewas similar with that of MA−Venus−H5N1 (FIG. 9). These data suggest thatthe PB2 and PA genes played a role in the pathogenicity of MA−Venus−H5N1virus in mice.

To assess the potential synergetic effects of the PB2 and PA genes onviral pathogenicity in mice, a reassortant carrying both the PB2 and PAgenes of MA−Venus−H5N1 [MA−(PB2+PA)] on the WT−Venus−H5N1 virus backboneand a reciprocal reassortant on the MA−Venus−H5N1 virus backbone[designated WT+MA−(PB2+PA) and MA+WT−(PB2+PA)] were rescued, andassessed for virulence in mice. The substitution of the PB2 and PA genesfrom MA−Venus−H5N1 virus into WT−Venus−H5N1 virus significantly enhancedits virulence in mice, with an MLD₅₀ value of 3.2 PFU (FIG. 9), and alsoenhanced virus spread and replication in mice, similar to that ofMA−Venus−H5N1 virus, and vice versa. Given that a single mutation ispresent in PB2 and in PA after mouse adaptation, these data indicatethat V25A of PB2 and R443K of PA synergistically contribute to thevirulence of MA−Venus−H5N1 virus in mice.

When checking the Venus expression of the above reassortants in MDCKcells, it was found that the MA−PB2 gene markedly increased Venusexpression (FIG. 7). In addition, Venus expression of WT+MA−PB2 virus inthe lungs was also appreciably enhanced (FIG. 8). The other single-genesubstitutions including MA−PA did not affect Venus expression; however,the double substitution of MA−PB2 and MA−PA on the WT−Venus−H5N1 virusbackbone increased Venus expression in MDCK cells and in mouse lungcompared with those achieved by WT−Venus−H5N1 and WT+MA−PB2 virus (FIGS.7 and 8). These data indicate that V25A of the PB2 protein plays a vitalrole in the Venus expression of MA−Venus−H5N1 virus in vitro and invivo, and that R443K of the PA protein enhances the PB2 effect on Venusexpression.

The Amino Acid at Position 25 in the PB2 Protein Significantly EnhancesViral Replication in Mammalian Cells.

The replicative ability of these viruses was further examined in MDCKcells and it was found that the MA−Venus−H5N1 virus had similarreplicative capability with RG−MA virus and grew more efficiently thanWT−Venus−H5N1 virus, and that the titers of MA−Venus−H5N1 virus weresignificantly higher than those of WT−Venus−H5N1 virus at 36 and 48 hpi(FIG. 10). The contribution of the PB2 and PA viral segments to thereplication of the two viruses was then investigated. Significantlyhigher titers of WT+MA−PB2 and WT+MA−(PB2+PA) were observed comparedwith those of WT−Venus−H5N1 virus at several time points post-infection,yet the replication efficiency of WT+MA−PA was comparable to that ofWT−Venus−H5N1 virus (FIG. 10). The titers of WT+MA−(PB2+PA) were higherthan those of WT+MA−PB2 at 36 and 48 hpi although the difference was notstatistically significant (FIG. 10). These results indicate that theMA−PB2 gene enhances the replication of Venus−H5N1 virus in MDCK cells,and that this increase can be further enhanced by the presence of MA−PA,although MA−PA alone does not alter virus replication in MDCK cells.

The Mutations in the Polymerase Genes after Mouse Adaptation DecreaseViral Polymerase Activity in Mammalian Cells.

The polymerase activity of the viral ribonucleoprotein (RNP) complex hasbeen correlated with viral replication and virulence (Gabriel et al.,2005; Leung et al., 2010; Li et al., 2008; Salomen et al., 2006). Theactivity of the eight RNP combinations of PB1, PB2, and PA from eitherWT−Venus−H5N1 or MA−Venus−H5N1 virus was determined by measuringluciferase activity. The polymerase activity of the mouse-adapted viruswas near 4-fold less than that of WT−Venus−H5N1 virus (FIG. 11). Thesubstitution of any MA gene decreased the activity of the polymerasecomplex of WT−Venus−H5N1 virus, but the polymerase activity of complexescontaining the double substitution of MA−PB2 and MA−PA was significantlydecreased compared with that of WT−Venus−H5N1 virus and was similar withthat of MA−Venus−H5N1 virus. These results indicate that the polymeraseactivity of RNP complexes was notably decreased after mouse adaptation,which is not in agreement with the enhanced replication and virulence.

Molecular Determinants of Venus Stability in Venus−H5N1 Virus In Vitroand In Vivo.

To assess Venus stability in the WT−Venus−H5N1 and RG−MA viruses invitro, the two viruses were passaged five times in MOCK cells. Duringthese passages Venus-negative plaques were picked up from WT−Venus−H5N1virus, but not from RG−MA virus, suggesting that the Venus gene is morestable after mouse adaptation (Table 5). To identify the moleculardeterminants of this Venus stability, various reassortants were passagedfive times in MOCK cells. Venus-negative plaques were acquired fromreassortants with the MA−PB1, MA−NA, or MA−M gene, but we did not obtainany Venus-negative plaques from the fifth passages of Venus−H5N1 viruswith the MA−PB2, MA−PA, MA−(PB2+PA), or MA−NS gene (Table 5). These datasuggest that the MA−PB2, −PA, and −NS genes may play roles in Venusstability.

TABLE 5 Venus stability in Venus-H5N1 reassortants in MDCK cells^(a).No. of No. of No. of No. of Venus-negative passages in plaquesVenus-negative plaques after Virus MDCK cells checked plaques pickedrecheck WT-Venus-H5N1 2 73 5 4 3 111 5 1 4 79 5 0 5 61 12 2 RG-MA 2 66 10 3 144 8 0 4 73 1 0 5 75 2 0 WT + MA-PB2 5 84 2 0 WT + MA-PB1 5 126 4 1WT + MA-PA 5 104 16 0 WT + MA-(PB2 + PA) 5 123 6 0 WT + MA-NS 5 199 11 0WT + MA-NA 5 73 16 1 WT + MA-M ^(b) 5 69 10 10 ^(a)Each virus waspassaged five times in MDCK cells as describe in the Materials andMethods. Venus expression of different passage stocks was detected inMDCK cells by using fluorescence microscopy. Venus-negative plaques werepicked up and amplified in MDCK cells. Amplified Venus-negative plaqueswere rechecked for Venus expression to exclude false-negative plaques.^(b) 69 plaques of the fifth passage stock of WT + MA-M were checked byusing fluorescence microscopy, all of which were “Venus-negative”. Tenof these plaques were picked up to further confirm the lack of Venusexpression, all of which were confirmed to be Venus-negative.

To further evaluate the roles of these different genes on Venusstability, the NS segments of the fifth-passage stocks from differentreassortants were amplified by using PCR and NS-specific primers. Exceptfor the Venus-NS segment (1.9 kb), the deleted NS segments weredetectable, at a level similar to that for the NS segment of PR8, atless than 1 kb. The deleted NS segments of WT−Venus−H5N1 and of thereassortants with the MA−NA and MA−M genes were much brighter than thoseof the other reassortants (FIG. 12), further implying that the MA−NA andMA−M genes do not contribute to Venus stability in vitro. Although RG−MAvirus and the reassortants with MA−NS, MA−PA, or MA−PB2 were morestable, the deleted Venus-NS segments were still amplified by using PCRalbeit to a lesser degree (FIG. 12). The deleted NS segments from thevarious reassortants were extracted and sequenced, and the differentdeletion forms were identified from the different reassortants (FIG.13).

In addition, to examine Venus stability in vivo, B6 mice were inoculatedwith 105 PFU of WT−Venus−H5N1 virus, RG−MA virus, or WT+MA−(PB2+PA)virus. Lungs were collected on day 4 p.i., before the mice died, andwere homogenized in PBS. The supernatants were inoculated into MDCKcells, and at 48 hpi Venus-negative plaques were picked up and amplifiedin MDCK cells. It should be noted that sometimes the Venus signal of theplaque correlates with the condition of the cultured cells and thedetection time. Therefore the Venus expression of amplifiedVenus-negative plaques was rechecked in MDCK cells to exclude falsenegatives. More than 95 plaques were detected from each lung, and onlyone plaque without Venus expression was acquired from one of three miceinfected with RG−MA virus, twelve Venus-negative plaques from three miceinfected with WT+MA−(PB2+PA), and more than 15 Venus-negative plaquesfrom each mouse infected with WT−Venus−H5N1 virus (Table 6). Theseresults indicate that WT−Venus−H5N1 virus is the most unstable of theseviruses in vivo, and that the PB2 and PA genes from MA−Venus−H5N1 virusenhance Venus stability, albeit to a lesser extent than occurs inMA−Venus−H5N1 virus. The mutations on PB1, PB2, PA, and NS may thereforesynergistically contribute to Venus stability in MA−Venus−H5N1 virus invivo.

TABLE 6 Venus stability in Venus-H5N1 viruses in mice^(a). No. of No. ofNo. of Venus-negative Mouse plaques Venus-negative plaques after VirusNo. checked plaques picked recheck RG-MA 1# 105 4 1 2# 109 6 0 3# 127 50 WT + MA-(PB2 + PA) 4# 116 12 6 5# 111 8 3 6# 115 14 3 WT-Venus-H5N1 7#145 31 22 8# 120 27 18 9# 95 19 15 ^(a)Six-week-old SPF C57/BL6 micewere infected intranasally with 10⁵ PFU of each virus in a 50-μl volume.Three mice from each group were euthanized on day 4 p.i. and their lungtissues were collected and homogenized in PBS. The supernatants of thelung samples were inoculated into MDCK cells to check for Venusexpression, and Venus-negative plaques were picked up and amplified inMDCK cells. Amplified Venus-negative plaques were rechecked for Venusexpression to exclude false-negative plaques.

Discussion

Previously, a visualizable H5N1 virus expressing a Venus reporter genethat became more lethal to mice and more stable after mouse adaptationwas constructed (Example 1). In this study, the whole genome of thisvirus (MA−Venus−H5N1) was sequenced, and seven amino acids that differedfrom the WT−Venus−H5N1 virus sequence were identified. To explore themolecular determinants for the differences in virulence and Venusexpression in mice between these two viruses, a series of reassortantsof both viruses was generated using reverse genetics. The doublemutation of PB32 (V25A) and PA (R443K) was found to dramatically enhancethe pathogenicity of WT−Venus−H5N1 in mice. V25A of P1B2 alsosignificantly increased Venus expression and viral replication in MDCKcells and in mice, and that R443K of PA further enhanced these effects.The stability of different reassortants was examined in vitro, thereassortants with MA−PB2, MA−PA, or MA−NS were found to be more stable.These results suggest that the PB32 and PA proteins play roles in thepathogenicity and Venus stability of Venus-expressing H5N1 viruses inmammalian hosts.

The pathogenicity of highly pathogenic H5N1 avian influenza viruses inmammals is determined by multiple viral genes. For example, the HAprotein plays crucial roles in the systemic replication and lethalinfection of H5 subtype viruses in chickens (Kawaoka and Webster, 1988)and mammals (Hatta et al., 2001; Suguitan et al., 2012). The HA and NSgenes of H5N1 virus also contribute to high virulence in ferrets (Imaiet al., 2010). The NS1 protein helps to subvert the antiviral immuneresponse of the host and is essential for the pathogenicity of H5N1viruses in mice (Jiao et al., 2005). Mutations in the M1 protein alsoaffect the virulence of H5N1 viruses in mice (Fan et al., 2009). Theamino acids at position 627 and 701 of PB2 are key determinants of thehigh virulence of H5N1 influenza viruses in mammals (Hatta et al., 2001;Li et al., 2005). Lastly, the PA protein is reported to contribute tothe virulence of H5N1 avian influenza viruses in domestic ducks (Song etal., 2011) and in mice (Hu et al., 2013). Here, it was found that V25Aof PB2 and R443K of PA synergistically contribute the pathogenicity ofH5N1 virus in mice.

Based on all of the influenza virus sequences (23514 PB2 proteins and24240 PA proteins) available in the public database (www.fludb.org), itwas found that 25V in PB2 and 443R in PA are extremely conserved,whereas 25A in PB2 is present in only two viruses[A/Mallard/ON/499/2005(H5N1), accession number EF392844; andA/Zhejiang/92/2009(H1N1), accession number CY095997] and 443K in PA ispresent in only one strain, isolated from a quail[A/Quail/Shantou/1425/2001(H9N2), accession number EF154846]. Althoughthe virulence of these viruses in mice is unknown, the present study isthe first to suggest that the combination of 25A in PB2 and 443K in PAcontributes to the increased virulence of a virus in mice and is aunique feature of MA−Venus−H5N1 virus.

The RNA polymerase of influenza A virus consists of the PB1, PB2, and PAsubunits, and is implicated in numerous essential processes in the virallife cycle (Naffakh et al., 2005). PB1 performs polymerase andendonuclease activities, PB2 is responsible for capped-RNA recognition,and PA is involved in RNA replication and proteolytic activity (Obayasjoet al., 2005). The interfaces of these polymerase subunits are essentialfor transcription initiation (He et al., 2008; Sugiyama et al., 2009).Residues 1-37 at the N-terminus of the PB2 protein play a vital role inbinding to the PB1 protein and affect the RNA polymerase activity, andthese residues are highly conserved among all subtypes of influenzavirus (Sugiyama et al., 2009). The amino acid at position 25 of PB2 islocated within the third α-helix (amino acids 25 to 32) of itsPB1-binding domain (Sugiyama et al., 2009). In this study, the aminoacid at position 25 in PB2 was found to be changeable, and V25A in PB2was found to increase viral replication in mammalian cells and in mice,resulting in higher pathogenicity of the H5N1 virus in mice. The R443residue of the PA protein also plays a role in replication activity(Obayashi et al., 2008; Regan et al., 2006), and the mutation R443A inPA prevents the production of infectious virus (Regan et al., 2006). Inthis study, reassortants with R443K in their PA protein were rescued,and demonstrated that R443K in PA enhances viral replication in mouselungs, reinforcing it was the virulence of H5N1 virus in mice. Thepresent data thus further emphasize the role of the amino acid atposition 443 of the PA protein for influenza virus.

Earlier reports have shown that the polymerase activity of the viral RNPcomplex closely correlates with viral replication and virulence (Gabrielet al., 2005; Leung et al., 2010; Li et al., 2008; Salomon et al.,2006). Viruses with higher polymerase activity in mammalian cellsgenerally show higher virulence in mice (Zhang et al., 2014) and ferrets(Salomen et al., 2006). However, viruses with high polymerase activityare not always lethal to mice, which suggests that high pathogenicity ofa virus in its host may require a certain level of polymerase activity(Gabriel et al., 2005). In this study, it was found that MA−Venus−H5N1virus was more lethal to mice than was its wild-type counterpart, yet ithad much lower polymerase activity, and any RNP combination with apolymerase gene from MA−Venus−H5N1 also had lower activity. Theseresults may imply that the polymerase activity of the vRNP complexclosely correlates with the viral genome, and that the lower level ofpolymerase activity is more compatible with the reconstructed genome ofVenus−H5N1, which benefits its high pathogenicity in mice.

With the development of living imaging in vivo, the ability to visualizeinfluenza viruses carrying fluorescent reporter genes will be of greatbenefit influenza virus-related research (Heaton et al, 2013; Helft etal., 2012; Manicassamy et al., 2010; Pan et al., 2013; Example 1). Aneffective virus for this purpose should have good replicative abilityand show considerable pathogenicity in its host. Moreover, it shouldboth highly and stably express its fluorescent reporter protein. Manyattempts to construct influenza A viruses carrying the GFP reporter genehave been reported (Kittel et al., 2004; Manicassamy et al., 2010);however, some of these viruses showed low replication or poorpathogenicity in mice (Kittle et al., 2004), while some producedrelatively low fluorescent signals or did not stably express GFP duringvirus replication in vitro and in vivo (Manicassamy et al., 2010). Thepresent data demonstrate that not only is MA−Venus−H5N1 virus highlypathogenic to mice, but it also highly and stably expresses Venusfluorescent protein in vitro and in vivo. In the present analysis of themolecular determinants of Venus expression and Venus stability, it wasfound that V25A in PB2 played a role in determining Venus expression,which was further enhanced by the presence of R443K in PA. The analysisof Venus stability revealed that the single gene of MA−PB1, −PB2, −PA,or −NS determines Venus stability in vitro, but in vivo the situation ismore complex and mutations in PB1, PB2, PA, and NS may synergisticallycodetermine Venus stability in MA−Venus−H5N1 virus.

In summary, molecular determinants in a mouse-adapted Venus−H5N1 viruswere identified that play a crucial role in the pathogenicity of thevirus in mice, and in its Venus expression and Venus stability in vitroand in vivo. These molecular markers will benefit future research onanti-influenza virus drug and vaccine development.

Example III Materials and Methods

Cells and viruses. Madin-Darby canine kidney (MDCK) cells weremaintained in minimum essential medium (MEM) containing 5% of newborncalf serum (NCS). Human embryonic kidney 293T (HEK293T) and HEK293 cellswere maintained in Dulbecco's modified Eagle medium supplemented with10% fetal calf serum (FCS). A/Puerto Rico/8/34 (H1N1; PR8) (Horimoto etal., 2007) and each NS1−Venus PR8 virus were generated by using reversegenetics and were propagated in MDCK cells at 37° C. for 48 hours in MEMcontaining L-(tosylamido-2-phenyl) ethyl chloromethyl ketone(TPCK)-treated trypsin (0.8 μg/mL) and 0.3% bovine serum albumin (BSA)(Sigma Aldrich).

Adaptation of NS1−Venus PR8 virus in mice. Six- to eight-week-old femaleC57BL/6 mice (Japan SLC) were intranasally infected with 50 μL of2.3×10⁶ plaque-forming units (PFU) of NS1−Venus PR8 virus. Lungs wereharvested 3-6 days post-infection (dpi) and homogenized in 1 mL ofphosphate-buffered saline (PBS). To obtain a clone with highproliferative ability and Venus expression, plaque purification of thelung homogenate using MDCK cells was performed. A large, highlyVenus-expressing plaque was picked and the cloned virus was propagatedin MDCK cells at 37° C. for 48 hours, then 50 μL of the supernatant wasused as an inoculum for the next passage. These procedures were repeatedsix times.

Sequence analysis. Sequence analysis of viral RNA was performed asdescribed previously (Sakabe et al., 2011). Briefly, viral RNAs wereextracted by using a QIAamp Viral RNA mini kit (QIAGEN) and SuperscriptIII™ reverse transcriptase (Invitrogen) and an oligonucleotidecomplementary to the 12-nucleotide sequence at the 3′ end of the viralRNA (Katz et al., 1990) were used for reverse transcription of viralRNAs. Each segment was amplified by using PCR with Phusion High FidelityDNA polymerase (Finnzymes) and primers specific for each segment of thePR8 virus. The PCR products were purified and their sequences determinedby using ABI 3130xl (Applied Biosystems).

Plasmid construction and reverse genetics. Plasmids containing thecloned cDNAs of PR8 genes between the human RNA polymerase I promoterand the mouse RNA polymerase I terminator (referred to as Poll plasmid)were used for reverse genetics and as templates for mutagenesis. Themutations found in NS1-Venus PR8 virus after passage were introducedinto the plasmid constructs of PR8 by using site-directed mutagenesis(referred to as pPoIIR-PR8−PB2−E712D and pPolIR-PR8−HA−T380A,respectively). Reverse genetics was performed as described previously(Neumann et al., 1999). The eight Poll plasmids were cotransfected intoHEK293T cells together with eukaryotic protein expressing plasmids forPB2, PB1, PA, and NP derived from PR8 by using the TransIT-293transfection reagent (Mirus). Forty-eight hours after transfection, thesupernatant was harvested and propagated once in MDCK cells at 37° C.for 48 hours in MEM containing TPCK-treated trypsin (0.8 □g/mL) and 0.3%BSA. Cell debris was removed by centrifugation at 2,100×g for 20 minutesat 4° C., and the supernatants were stored at −80° C. until use. Thevirus titers were determined by means of a plaque assay using MDCKcells.

Polykaryon formation assay. Polykaryon formation assay was performed asdescribed previously (Imai et al., 2012) with modifications. HEK293cells propagated in 24-well plates were infected with wild-type PR8 orPR8 possessing the hemagglutinin (HA) mutation found in NS1−Venus PR8 MAvirus in DMEM containing 10% FCS at a multiplicity of infection (MOI) of10. At 18 hours post-infection, cells were washed with MEM containing0.3% BSA and treated with TPCK-treated trypsin (1 μg/mL) in MEMcontaining 0.3% BSA for 15 minutes at 37° C. to cleave the HA on thecell surface into HA1 and HA2. Trypsin was inactivated by washing thecells with DMEM containing 10% FCS. To initiate polykaryon formation,cells were exposed to low-pH buffer (145 mM NaCl, 20 mM sodium citrate(pH 6.0-5.4)) for 2 minutes at 37° C. Then the low-pH buffer wasreplaced with DMEM containing 10% FCS and the cells were incubated for 2hours at 37° C. The cells were then fixed with methanol and stained withGiemsa's solution. A microscope mounted with a digital camera (Nikon)was used to obtain photographic images.

Western blotting. MDCK cells were infected with each virus at an MOI of1 without trypsin. The cells were lysed with Novex® Tris-Glycine SDSsample buffer (Invitrogen) 12 hours after infection and subjected toSDS-polyacrylamide gel electrophoresis. Then, the proteins weretransferred to a PVDF membrane in transfer buffer (100 mM Tris, 190 mMglycine). After membrane blocking, the membranes were incubated with arabbit anti-GFP polyclonal antibody (MBL) or rabbit antiserum toA/WSN/33(H1N1)(R309), which was available in our laboratory. Thisantiserum reacts with influenza viral proteins including HA, NP, andmatrix protein (M1). After incubation with the primary antibodiesfollowed by washing with PBS containing 0.05% Tween-20 (PBS-T), themembranes were incubated with ECL™ anti-rabbit IgG HRP-linked wholeantibody (GE Healthcare). Finally, specific proteins were detected byusing the ECL Plus Western Blotting Detection System (GE Healthcare).The VersaDoc Imaging System (Bio-Rad) was used to obtain photographicimages.

Pathogenicity and replication of viruses in mice. Six-week-old femaleC57BL/6 mice were intranasally infected with 50 μL of 10³, 10⁴ or 10⁵PFU of each virus. Four mice per group were monitored for survival andbody weight changes for 14 days after infection. Three mice per groupwere infected with 10³ PFU of each virus and euthanized on the indicateddays. Their lungs were collected to determine viral titers by means ofplaque assay on MDCK cells.

Immunofluorescence assay. Six-week-old female C57BL/6 mice wereintranasally infected with 50 μL of 10⁴ PFU of each virus. Three miceper group were euthanized on the indicated days. To fix the lungs, theywere intratracheally injected with 800 μL of 4% paraformaldehyde (PFA)phosphate buffer solution and then removed. After incubation with 10 mLof 4% PFA at 4° C. for 4 hours, the buffer was replaced with 10%, 20%,and 30% sucrose in PBS in a stepwise fashion. Then lungs were embeddedin Optimum Cutting Temperature (OCT) Compound (Tissue-Tek) and frozen inliquid nitrogen. Frozen sections (6 μm in thickness) were permeabilizedin 0.2% Triton X-100 in PBS and incubated with primary antibodies at 4°C. for 12 hours. Primary antibodies were goat anti-Clara cell 10 kDaprotein (CC10) (Santa Cruz, sc-9772), rabbit anti-surfactant protein C(SP-C) (Santa Cruz, sc-13979), golden Syrian hamster anti-podoplanin(eBioscience, eBio8.1.1), and rabbit anti-calcitonin gene-relatedpeptide (CGRP) (Sigma-Aldrich, C8198). After being washed with PBS, thesections were incubated with species-specific fluorescencedye-conjugated secondary antibodies at room temperature for 30 minutes.Nuclei were stained with Hoechst33342 (Invitrogen). A Nikon A1 confocalmicroscope (Nikon) was used to observe the sections.

Preparation of transparent samples. Transparent samples were prepared byusing SCALEVIEW A2 (Olympus) in accordance with a previous report (Hamaet al., 2012). Six-week-old female C57BL/6 mice were intranasallyinfected with 50 μL of 105 PFU of each virus. Intracardial perfusion wasperformed on the indicated days and lungs were fixed with 4% PFA in PBSfor 4 hours at 4° C. Lungs were incubated with 10%, 20%, and 30% sucrosein PBS as described above, embedded in OCT compound, and frozen inliquid nitrogen. After the samples were thawed and rinsed in PBS, theywere fixed again with 4% PFA in PBS for 30 minutes at room temperature.Then the lungs were transferred to SCALEVIEW A2 and incubated at 4° C.for at least 2 weeks. SCALEVIEW A2 was exchanged every 2-3 days.Transparent samples were observed by using a stereo fluorescencemicroscope (Leica M205FA) mounted with a digital camera (DFC365FX) andfilter GFP 3 (480/40 LP510).

Flow cytometry. To prepare single-cell suspensions, lungs were mincedwith scissors and digested with 20 mg of collagenase D (Roche) and 200units of DNase (Worthington) for 30 minutes at 37° C. Samples were thenpassed through 100-sm cell strainers and red blood cells were lysed byred blood cell lysis buffer (Sigma Aldrich). Single-cell suspensionswere stained with a combination of the following antibodies:allophycocyanine-conjugated anti-F4/80 (eBioscience, BM8),allophycocyanine-cyanine 7-conjugated anti-CD11b (BioLegend, M1/70),phycoerythrin-cyanine 7-conjugated anti-CD11c (BD PharMingen, HL3), andeFluor 450-conjugated CD45 (eBioscience, 30-F11). Dead cells werestained with via-probe (Becton Dickinson). Stained samples were analyzedwith FACSAria II (Becton Dickinson and Company) and FlowJo software(TreeStar).

RNA isolation and integrity. Venus-positive and -negative cells fromthree pooled lungs were collected in TRIzol Reagent (Invitrogen). TotalRNA was extracted by isopropanol precipitation with glycogen as acarrier. Isolated total RNA integrity was assessed by determining UV260/280 absorbance ratios and by examining 28S/18S ribosomal RNA bandswith an Agilent 2100 bioanalyzer (Agilent Technologies) according to themanufacturer's instructions.

Microarray analysis. Forty nanograms of total RNAs was amplified byusing the Arcturus® Riboamp® Plus RNA Amplification Kit (Lifetechnologies). Cy3-labeled complementary RNA probe synthesis wasinitiated with 100 ng of total RNA by using the Agilent Low Input QuickAmp Labeling kit, one color (Agilent Technologies) according to themanufacturer's instructions. The Agilent SurePrint G3 Gene Mouse GE 8×60K microarray was also used. Slides were scanned with an Agilent'sHigh-Resolution Microarray Scanner, and image data were processed byusing Agilent Feature Extraction software ver. 10.7.3.1. All data weresubsequently uploaded into GeneSpring GX ver 12.5 for data analysis. Forthe data analysis, each gene expression array data set was normalized tothe in silico pool for samples from mice inoculated with PBS.Statistically significant differences in gene expression between theVenus-positive cells and -negative cells were determined by usingone-way analysis of variance (ANOVA) followed by the Turkey HSD post-hoctest (P<0.05) and the Benjamin-Hochberg false discovery rate correction.Differentially expressed genes were further filtered to include geneswhose expression changed 2.0-fold relatively to the level in the PBSgroup. Genes that passed the statistical analysis were further assignedto a gene ontology (GO) grouping.

Results

Establishment of a mouse-adapted NS1−Venus PR8 virus. Although NS1−VenusPR8 WT virus was successfully rescued by reverse genetics, this viruswas avirulent in mice (MLD₅₀: >10⁵ PFU), and the expression of Venus wasvery weak in MDCK cells and in the lung sections of mice infected withthis virus. To increase the virulence and Venus expression of NS1−VenusPR8 WT virus, the virus was serially passed in mice via intranasalinfection with plaque-purified high Venus-expressing clones (seeExamples I and II). After six serial passages, the virulence of thevirus appeared to have increased; therefore, this mouse-adaptedNS1−Venus PR8 WT virus was sequenced to look for mutations.

The sequence analysis revealed that two amino acid substitutions hadoccurred after passaging (Table 7).

TABLE 7 Amino acid substitutions in NS1-Venus PR8 MA virus. amino acidencoded Protein amino acid position PR8 NS1-Venus PR8 MA PB2 712 E D HA380 T AOne of the mutations was in PB2 (a glutamine acid-to-asparagine acidsubstitution at position of 712), and the other was in HA (athreonine-to-alanine substitution at position of 380). To confirm theircontribution to pathogenicity in mice, these mutations were introducedinto a correspondent poll plasmid, and reverse genetics used to generateNS1−Venus PR8, which possessed the two mutations (referred to asNS1−Venus PR8 MA virus). The pathogenicity of NS1−Venus PR8 MA virus washigher than that of NS1−Venus PR8 WT virus (MLD₅₀: 2.1×10W PFU).Furthermore, the Venus signal in the lungs from mice infected withNS1−Venus PR8 MA virus was strong, whereas in the lung infected withNS1−Venus PR8 WT and that infected with PR8, no Venus signal wasdetected (data not shown). NS1−Venus PR8 MA, therefore, showed promiseas a useful reporter virus.

Comparison of mutant virus replication in MDCK cells. To compare thegrowth of these viruses in a cell line, two single-gene reassortantswere generated that possessed the PB2 or HA gene of NS1−Venus PR8 MAvirus and the remaining genes from NS-Venus PR8 WT virus for use inexperiments with the NS1-Venus PR8 WT and NS1−Venus PR8 MA viruses. MDCKcells were infected with these viruses at an MOI of 0.001 and viraltiters in supernatants were determined every 12 hours by means of aplaque assay (FIG. 14). Although NS1−Venus PR8 WT virus grew to 10^(6.5)PFU/mL, NS1−Venus PR8 MA virus grew to more than 10⁸ PFU/mL, comparableto wild-type PR8 virus. While the viral titers of NS1−Venus PR8 PB2virus and NS1-Venus PR8 HA virus reached approximately 1075 PFU/mL,these were lower than that of NS1−Venus PR8 MA virus. Therefore, thegrowth capability of NS1−Venus PR8 MA virus was remarkably improved inMDCK cells, and the mutations in the PB2 and HA genes acted in anadditive manner.

Comparison of the pathogenicity and replication in mice of the mutantviruses. Next, to assess their pathogenicities, C57BL/6 mice wereinfected with 10⁵, 10⁴ or 10³ PFU of these viruses and monitored theirbody weights and survival (FIG. 15). The body weights of the miceinfected with 10⁵ PFU of these viruses dramatically decreased and 1 outof 4 mice infected with NS1−Venus PR8 WT virus and all of the miceinfected with NS1−Venus PR8 PB2 and NS1−Venus PR8 MA virus had to beeuthanized during the observation period. In addition, mice infectedwith 10⁴ PFU of NS1−Venus PR8 PB2 and NS1−Venus PR8 MA virus showedpronounced body weight loss, and 1 out of 4 mice infected with NS1−VenusMA virus and 2 out of 4 mice infected with NS1−Venus PR8 PB2 virussuccumbed to their infection. On the other hand, although the bodyweights of the mice infected with 10⁴ PFU of NS1−Venus PR8 HA andNS1−Venus PR8 WT virus decreased slightly, all of the mice survived. Inthe case of infection with 10³ PFU, while the body weights of the miceinfected with NS1−Venus PR8 PB2 and NS1−Venus PR8 MA decreased slightly,all of these mice also survived. Mice infected with 10³ PFU of NS1−VenusPR8 WT and NS1−Venus PR8 HA showed little body weight loss, and all ofthe mice survived. The viral titers of these viruses were determined inmouse lung (FIG. 16). Mice were infected with 10³ PFU of the viruses andlungs were collected on days 3, 5, and 7 after infection. The maximumvirus lung titer from mice infected with NS1−Venus PR8 PB2 virus was>10⁶ PFU/g, which was similar to that from mice infected with NS1−VenusPR8 MA virus. In contrast, virus titers in lungs from mice infected withNS1−Venus PR8 WT and NS1−Venus PR8 HA virus were significantly lowerthan those in lungs from mice infected with NS1−Venus PR8 PB2 andNS1−Venus PR8 MA virus at all time points. Finally, viruses were notdetected in lungs from mice infected with NS1−Venus PR8 WT at 7 daysafter infection. Taken together, these results demonstrate that only thePB2 mutation affected the pathogenicity and replication of NS1−Venus PR8MA virus in mice.

The stability of Venus expression by NS1−Venus PR8 MA virus duringreplication in vitro and in vivo. In the Manicassamy study (Manicassamyet al., 2010), the proportion of GFP-negative virus increased over time.This is one of the obstacles to utilizing this virus for live imagingstudies. The stability of Venus expression by NS1−Venus PR8 MA virus wasassessed during replication in MDCK cells (FIG. 17A). More than 90% ofplaques were Venus-positive even 72 hours after infection. The positiverate of Venus expression was monitored during repeated passages of thevirus in cell culture (FIG. 17B). Approximately 90% of plaques expressedVenus even after 5 passages, suggesting that Venus expression byNS1−Venus PR8 MA virus was stable in cell culture. Finally, Venusexpression was confirmed to be stable during virus replication in vivo(FIG. 17C). A plaque assay was performed using lung homogenates andestimated the positive rate of Venus expression essentially as describedabove. Although the percentage of Venus-positive plaques was more than85% at 3 days after infection, that of Venus-positive plaques wasapproximately 75% at 7 days after infection. Taken together, theseresults indicate that Venus expression by NS1−Venus PR8 MA virus isstable during replication in vitro, and the percentage of Venus-positiveplaques in mouse lung was similar to that reported previously(Manicassamy et al., 2010).

The PB2−E712D substitution is responsible for high Venus expression. TheVenus expression level of NS1−Venus PR8 MA virus was substantiallyhigher than that of NS1−Venus PR8 WT virus. Since PB2 is one of thesubunit of the influenza virus polymerase, it was hypothesized that thePB2−E712D substitution was important for the augmentation of Venusexpression. To compare the Venus protein expression, western blots ofthe viral protein and Venus in infected cells were performed (FIG. 18A).Twelve hours post-infection, although the amount of M1 protein wassimilar for all of the viruses, the amount of Venus protein was higherin cells infected with NS1−Venus PR8 PB2 and NS1−Venus PR8 MA viruscompared with the other two viruses that possessed the parental PB2gene. Venus expression in infected cells was also observed by using aconfocal laser microscope (FIG. 18B). As expected, the Venus signals inthe cells infected with NS1−Venus PR8 PB2 and NS1−Venus PR8 MA viruswere stronger than in the cells infected with the two viruses thatpossessed parental PB2 gene. Taken together, these results demonstratethat the PB2−E712D substitution was responsible for the high Venusexpression.

To demonstrate that the PB2−E712D mutation increased the Venusexpression levels, MDCK cells were infected with the indicated virusesat an MOI of 1 and performed confocal microscopy 12 hours later (FIG.18C). As expected, the levels of the NS1−Venus fusion protein werehigher in cells infected with MA−Venus−PR8 or PB2-Venus−PR8 than inthose infected with WT−Venus−PR8 or HA-Venus−PR8 (FIG. 18C).

Collectively, the data indicate that the PB2−E712D substitution isprimarily responsible for the increased replicative ability, Venusexpression, and virulence in mice of MA−Venus−PR8 virus. To assesswhether the PB2−E712D mutation directly affects the viral polymeraseactivity in a minireplicon assay, HEK293 cells were transfected withviral protein expression plasmids for NP, PA, PB1, and PB2 or PB2−E712D,together with a plasmid expressing a vRNA encoding the fireflyluciferase gene; the pRL-null luciferase protein expression plasmid(Promega) served as a transfection control. Luciferase activities weremeasured by using a Dual-Glo luciferase assay system (Promega) at 48hours post-transfection (Ozawa et al., 2007). Unexpectedly, thepolymerase activity of PB2−E712D was lower than that of the parental PB2(FIG. 18D). Similar results were obtained with canine MDCK cells (datanot shown). In the context of a minireplicon that measures viralreplication and transcription, the PB2−E712D mutation is thusattenuating; in contrast, this mutation enhances viral growth in thecontext of replicating virus. These findings indicate that the PB2protein functions not only in viral replication/transcription, butperforms additional roles in the viral life cycle.

The HA−T380A substitution raises the threshold for membrane fusion. TheHA vRNA of MA−Venus−PR8 did not significantly increase the virulence ofWT−Venus−PR8 in mice; however, HA-Venus−PR8 virus grew more efficientlyin MDCK cells than WT−Venus−PR8 (FIG. 14), suggesting a contribution ofthe HA-T380A mutation to, at least, virus replication in cultured cells.Because the HA−T380A substitution is located on an a-helix in the HA2subunit (Gamblin et al., 2004), its effect on HA membrane-fusionactivity was evaluated by using a polykaryon formation assay (Imai etal., 2012). Briefly, HEK293 cells were infected with WT−PR8 or a mutantPR8 virus encoding HA−T380A at an MOI of 10. Eighteen hours later, cellswere treated with TPCK-treated trypsin (1 μg/mL) for 15 minutes at 37°C., exposed to low-pH buffer (145 mM NaCl, 20 mM sodium citrate (pH6.0-5.4)) for 2 minutes, incubated for 2 hours in maintenance medium at37° C., fixed with methanol, and stained with Giemsa's solution. Thewild-type HA had a threshold for membrane fusion of pH 5.5, whereas thethreshold for HA−T380A was pH 5.8 (FIG. 19), leading to theconformational change in HA at an earlier stage of endosome maturationduring influenza virus entry (Lozach et al., 2011). Changes in the pHthreshold for membrane fusion may affect HA thermostability (Ruigrok etal., 1986), an effect that we did not observe at 50° C. (data notshown).

Time-course observation of virus propagation in whole mouse lung.NS1−Venus PR8 MA virus allows the observation of virus-infected cellswithout immunostaining because the Venus expression by this virus issufficiently high to permit the visualization of infected cells with amicroscope. To observe how influenza virus propagates in the lung,transparent lungs are treated with SCALEVIEW A2, a reagent that makesamples optically transparent without decreasing fluorescence intensitywere used (FIG. 20). Mice were intranasally infected with 105 PFU ofPR8, NS1−Venus PR8 WT, and NS1−Venus PR8 MA virus, and lungs werecollected on days 1, 3, and 5 after infection. After treatment withSCALEVIEW A2, the samples were observed using a stereo fluorescencemicroscope. Venus signals that were directly observed were ambiguousbecause of insufficient transparency. Therefore, the transparent sampleswere dissected in the direction of the long axis to expose the bronchi(FIG. 20, lower panel, “cut”). Venus expression was not observed in thetransparent samples from mice infected with NS1−Venus PR8 WT virus atany time point (FIGS. 20G and H). Samples collected at 3 dayspost-infection are shown. In the case of NS1−Venus PR8 MA virus-infectedlungs, although Venus signals were not observed at 1 day post-infection(FIGS. 20A and B), Venus expression was clearly observed in a largeportion of the epithelial cells of the bronchi at 3 days post-infection(FIGS. 20C and D). Venus expression was also occasionally observed inalveolar epithelial cells around the bronchus. At 5 days post-infection,most of the Venus-positive cells found in the bronchial epithelium haddisappeared and the number of Venus-positive cells in the bronchiole andalveoli had increased (FIGS. 20E and F). On the basis of theseobservations, it may be that the Venus-positive cells found in thebronchi at 3 days post-infection died and the influenza virus spreadfrom the bronchi to the bronchioles and alveoli over time. No obviousVenus signals were observed in the transparent lungs from the miceinoculated with PR8 or PBS (FIGS. 201-L). These results demonstrate thatNS1−Venus PR8 MA virus and transparent reagent SCALEVIEW A2 permit thevisualization of the dynamics of influenza virus infection in whole lunglobes.

Identification of the target cells of NS1−Venus PR8 MA virus in mouselung. Transparent lungs infected with NS1−Venus PR8 MA virus revealedthat influenza virus first infected the bronchial epithelium andsubsequently invaded the alveoli over time. Next, to identify the targetcells of NS1−Venus PR8 MA virus, an immunofluorescence assay of frozensections was performed using several antibodies specific for lung cells(FIG. 21). The epithelial cells of the bronchi and bronchioles includeClara cells, ciliated cells, goblet cells, and a small number ofneuroendocrine cells, whereas alveoli comprise type I and type IIalveolar epithelial cells. Of these cell types, I focused on Clara cellsand type II alveolar epithelial cells because Clara cells constitute thebulk of the lumen of bronchi and bronchioles (Rawlins et al., 2006), andtype II alveolar epithelial cells have previously been reported to be atarget of influenza virus (Baskin et al., 2009). At 3 dayspost-infection, a large proportion of the bronchiole cells wereVenus-positive and almost all of these cells were CC10-positive (FIG.21A). In addition, cuboidal Venus signals in the alveolar regions weremerged with SP-C positive cells (FIG. 21B, white arrowheads). Althoughrare, Venus-positive type I alveolar epithelial cells were observed at 5days post-infection (FIG. 21B, white arrow). However, Venus expressionin neuroendocrine cells was never detected (data not shown).

Flow cytometry was performed to determine whether alveolar macrophagesand monocytes were infected with NS1−Venus PR8 MA virus, because theseimmune cells are present in lung and function as the first line ofdefense against inhaled microbes and particulates. Alveolar macrophageswere distinguished monocytes on the basis of the CD11b expression levelin the F4/80⁺ population (FIG. 22A). Mice were infected with 105 PFU ofPR8 or NS1−Venus PR8 MA virus and the total number of these cells werecompared. After influenza virus infection, although the number ofalveolar macrophages was rarely different from that of the controlgroup, the number of monocyte dramatically increased because monocytesinfiltrated sites of infection from blood vessels (FIGS. 22B and C). Asto the proportion of Venus-positive cells, 3.16%±0.59% of the alveolarmacrophages were Venus-positive cells and 1.55%±0.07% of the monocyteswere Venus-positive at 3 days post-infection (FIGS. 22D and E). Further,the number of Venus-positive cells decreased slightly between 3 days and5 days after NS1−Venus PR8 MA virus infection. For the PR8 infection,the number of Venus-positive cells was comparable to that inmock-treated mice. Taken together, these results demonstrate that theClara cells in the bronchus and bronchiolus, type II alveolar epithelialcells, monocytes, and alveolar macrophages in the alveolar regions ofthe lung are target cells of influenza virus.

Differential gene expressions between Venus-positive and -negative cellsin the F4/80⁺ cell population. Because alveolar macrophages andmonocytes act as the first line of defense against inhaled microbes, itis possible that infection of these cells with influenza virus mightinfluence their ability to prevent the spread of infection. To assessthis, the gene expression profiles between the Venus-positive and-negative cells among the alveolar macrophage and monocyte populationswere compared by means of microarray analysis. Because the number ofVenus-positive alveolar macrophages and monocytes that could becollected from one mouse by using flow cytometry was too small toperform a microarray analysis, these cells were analyzed together asF4/80⁺ cells and pooled from three mice. Live mononuclear cells weregated as CD45⁺ and via-probe⁻ cells. As shown in FIG. 22A, the cellswere confirmed as alveolar macrophages and monocytes on the basis ofCD11b expression levels in the F4180⁺ population. Venus-positive and-negative F4/80 cells were sorted from a fraction of the livemononuclear cells by FACSAria II. Since CD11c^(high) alveolarmacrophages possess high autofluorescence, the possibility existed foroverlap with the Venus signal. Therefore, CD11c^(high) alveolarmacrophages with intermediate expression of Venus were excluded from theVenus-positive fraction (FIG. 23A). From confocal microscopicobservation of the sorted cells, these cells could be collected properlybased on Venus expression (FIG. 23B). In addition, given that Venusexpression was observable throughout the cell, these cells would havebeen infected with virus, but did not engulf the infected cells. Themicroarray analysis revealed thousands of genes whose expressionstatistically changed at least 2.0-fold relative to the level of F4/80⁺cells from mice inoculated with PBS (data not shown). Among these genes,633 genes whose expression statistically differed by at least 4.0-foldbetween Venus−positive and -negative F4/80⁺ cells were identified (FIG.24A). Gene Ontology analysis revealed that these genes were involved inextracellular activity (FIG. 248). For genes annotated in “cytokineactivity,” a total of 24 genes had changed expression levels, includingseveral cytokines, such as type I interferon (IFN), and chemokines (FIG.24C). All of these genes except for the genes for interleukin (IL)-4 andCxcl13 [chemokine (C—X—C motif) ligand 13] were up-regulated inVenus-positive cells relative to Venus-negative cells. Moreover, when Ifocused on the genes annotated in “response to wounding”, most genesincluding those for collagen type 1α1 (Col1a1), collagen type 3α1(Col3a1), collagen type 5α1 (Col5a1), hyaluronoglucosamidase 1 (Hyal1),and fibrinogen γ chain (Fgg) were up-regulated in Venus-positive F4/80⁺cells (FIG. 24D). Taken together, these results demonstrate that a smallnumber of cells relative to the total number of F4/80⁺ cells wasinfected with influenza virus and that the gene expression levels ofseveral cytokines and chemokines were enhanced in the virus-infectedcells at the site of infection. Furthermore, F4/80⁺ cells infected withNS1−Venus PR8 MA virus enhanced the expression of genes involved in theresponse to wounding which would be caused by infection andinflammation.

Example IV

A vector is described above that can express a heterologous gene productfrom a fusion construct with the viral NS1 protein (FIG. 26). Inparticular, a PB2−E712D mutation stabilized expression of a heterologousgene product. The test virus, WT−Venus−PR8, was serially passaged toidentify other mutations in the polymerase complex which contribute tostabilization (FIG. 27).

Example V

An E-to-D mutation at position 712 of the polymerase subunit PB2(PB2−E712D) stabilized the inserted Venus gene (Fukuyama et al., 2015;Katsura et al., 2016). Also, a H5N1 virus carrying the Venus gene, whichwas inserted into the NS segment from PR8 (Venus−H5N1), was prepared(Fukuyama et al., 2015). Although, like WT−Venus−PR8, WT−Venus−H5N1showed moderate virulence and low Venus expression, we acquired avariant that became more lethal to mice and stably expressed Venus aftermouse adaptation. A V-to-A mutation at position 25 of the polymerasesubunit PB2 and a R-to-K mutation at position 443 of the polymerasesubunit PA contributed to the stable maintenance of the Venus gene (Zhaoet al., 2015). These results indicated that the composition of the viralpolymerase plays a role in the stabilization of the inserted foreigngene. However, the mechanisms by which the Venus gene can be deleted andhow polymerase mutations stabilize the Venus gene have remained unknown.

As disclosed below, the mechanisms of Venus gene stabilization wereinvestigated by comparing events upon infection with WT−Venus−PR8 andVenus−PR8 possessing the PB2−E712D mutation (Venus−PR8−PB2−E712D).Polymerase fidelity and RNA and protein expression in infected cellswere examined, and sequencing analysis coupled with coinfectionexperiments were performed to determine how the Venus gene is deleted.Moreover, additional mutations that contribute to the stabilization ofthe Venus gene were identified to further the understanding of thestabilization mechanisms.

Materials and Methods

Cells and viruses. Madin-Darby canine kidney (MDCK) cells were culturedin minimal essential medium (Gibco) with 5% newborn calf serum at 37° C.in 5% CO₂. Human embryonic kidney 293T (HEK293T) cells were cultured inDulbecco's modified Eagle medium supplemented with 10% fetal calf serum.WT-Venus−PR8 and Venus−PR8 mutants with NS segments encoding the Venusfluorescent protein (Fukuyama et al., 2015) were generated by usingreverse genetics (Neumann et al., 1999) and propagated in MDCK cells at37° C.

Venus stability. MDCK cells were infected with WT−Venus−PR8 or eachVenus−PR8 mutant at an MOI of 0.001. The supernatants were collected at48 h postinfection and titrated by using plaque assays in MDCK cells.Obtained viruses were similarly passaged four times. The proportion ofVenus-expressing plaques in virus stocks from different passages wasdetermined in MDCK cells by observing more than 65 plaques in each virusstock using fluorescence microscopy. To exclude false-positive plaques,Venus-negative plaques were picked up, amplified in MDCK cells, andreassessed for Venus expression.

Deep sequencing analysis WT−PR8, PR8−PB2−E712D, PR8−PB1−V43I (Cheung etal., 2014; Naito et al., 2015), and PR8−PB1−T123A (Pauly et al., 2017)were generated by reverse genetics (Neumann et al., 1999), and MDCKcells were infected at an MOI of 0.001. The supernatants were collectedat 48 h postinfection and titrated by using plaque assays in MDCK cells.The obtained viruses were passaged five times in the same way. Virus RNAwas extracted from viruses before passaging and from viruses passagedfive times by using a QIAamp viral RNA minikit (Qiagen). Reversetranscription-PCR (RT-PCR) was performed by using a Superscript IIIhigh-fidelity RT-PCR kit (Invitrogen). DNA amplicons were purified by0.45× of Agencourt AMpure XP magnetic beads (Beckman Coulter), and 1 ngwas used for barcoded library preparation with a Nextera XT DNA kit(Illumina). After bead-based normalization (Illumina), libraries weresequenced on the MiSeq platform in a paired-end run using the MiSeq v2,300 cycle reagent kit (Illumina). The raw sequence reads were analyzedby using the ViVan pipeline (Isakov et al., 2015). Here, a cutoff of 1%as the minimum frequency was used. Moreover, we defined an empiricalcutoff for the minimum read coverage: for a variant with 1% frequency,at least 1,000 reads should cover that region. Likewise, for a variantwith 0.1% frequency, 10,000 reads should cover that region. Namely, ifthe coverage was <1,000/(frequency), the variant was removed. Thesequencing data of the five-times passaged viruses were compared tothose of the viruses before passaging. The number of nucleotidemutations that were not observed before passaging but observed onlyafter passaging was counted. The number of mutations per nucleotide wascalculated for each segment and the mean values for all eight segmentsin each virus were compared.

Quantitative real-time PCR. MDCK cells were infected with WT−Venus−PR8or Venus−PR8−PB2−E712D at an MOI of 1 or mock infected with medium. Thetotal RNA was extracted from cells at 9 h postinfection by using anRNeasy minikit (Qiagen). Quantification of RNA was performed asdescribed previously (Kawakami et al., 2011). The primers for IFN-β, NSvRNA, NP vRNA, and β-actin were described previously (Kawakami et al.,2011; Kupke et al., 2018; Park et al., 2015). Data were analyzed withthe 2^(−ΔΔCT) method (Livak et al., 2001) and normalized to theexpression of β-actin mRNA.

Western blotting. MDCK cells were infected with each virus at an MOI of1 or mock infected with medium. Cells were lysed at the indicated timepoints with Tris-glycine SDS sample buffer (Invitrogen). The celllysates were sonicated, heated for 10 min at 95° C., and then subjectedto SDS-PAGE. SDS-PAGE was performed on Any kD Mini-PROTEAN TGX precastprotein gels (Bio-Rad). Proteins on SDS-PAGE gels were transferred to apolyvinylidene fluoride membrane (Millipore) and detected by using theindicated primary antibodies (rabbit anti-NS1 [GeneTex], mouseanti-Aichi NP [2S 347/4], mouse anti-R-actin [Sigma-Aldrich]), followedby secondary antibodies (sheep horseradish peroxidase [HRP]-conjugatedanti-mouse IgG [GE Healthcare] or donkey HRP-conjugated anti-rabbit IgG[GE Healthcare]). Signals of specific proteins were detected by usingECL Prime Western blotting detection reagent (GE Healthcare). Imageswere captured with a ChemiDoc Touch imaging system (Bio-Rad) andquantified by using Image Lab software (Bio-Rad). Coinfection analysis.Three nucleotides in the 3′ or 5′ region, which does not overlap thepackaging signal sequence (Fujii et al., 2005) of the NS segment ofWT−Venus−PR8, were substituted synonymously. These modified viruses wereused to coinfect MDCK cells at an MOI of 0.001 each or an MOI of 5 each.The supernatant was collected at 2 days or 8 h postinfection,respectively, and infected to MDCK cells. Venus−negative plaques werepicked up and amplified in MDCK cells, and then the sequences of the NSsegments in the obtained viruses were analyzed.

Identification of additional mutations that stabilize the Venus gene.WT−Venus−PR8 was infected to MDCK cells at an MOI of 0.001. Thesupernatant was collected at 2 days postinfection and infected to MDCKcells. Next, Venus-positive plaques were picked up and amplified in MDCKcells repeatedly until mutants stably expressing Venus fluorescence wereobtained. The sequences of the mutants were analyzed to identify aminoacid mutations in PB2, PB1, and PA. To determine whether these mutationscontributed to the stability of the Venus gene, mutants containing eachof the identified amino acid mutations were generated by reversegenetics (Neumann et al., 1999), and the Venus stability of each mutantwas examined as described above. Confirmed amino acid positions wereplotted on the crystal structure of the influenza virus polymerasecomplex (PDB ID 4WSB) by using the PyMOL molecular graphics system. InFIG. 32B, the polymerase internal tunnels were visualized by using theMOLEonline web interface (Pravda et al., 2018), and the information wasdeposited in ChannelsDB (Pravda et al., 2018). The percentage of strainsthat contained the identified amino acid was determined by using the“Sequencing Feature Variant Type” tool in the Influenza ResearchDatabase (Zhang et al., 2017; Noronha et al., 2012).

Statistical analysis. Statistically significant differences betweenWT−Venus−PR8 and Venus−PR8−PB2−E712D were assessed by using a two-tailedunpaired Student t test. A P value of <0.05 was considered significantlydifferent.

Results

Loss of Venus expression in WT−Venus−PR8 restores replicationefficiency. WT−Venus−PR8 and Venus−PR8−PB2−E712D were prepared by usingreverse genetics as previously described (Neumann et al., 1999). Thegene of the Venus fluorescent protein was inserted into the NS segmentas illustrated in FIG. 28A (Fukuyama et al., 2015). First, it wasconfirmed how quickly Venus expression was lost in WT−Venus−PR8 and therelationship between Venus deletion and virus titer. The viruses werepassaged in MDCK cells at a multiplicity of infection (MOI) of 0.001 andthe proportion of Venus-positive plaques measured (FIG. 288). It wasconfirmed that the expression of Venus was lost immediately inWT−Venus−PR8, whereas all plaques of Venus−PR8−PB2−E712D showed Venusexpression after four passages. Although WT−Venus−PR8 showed a lowertiter than Venus−PR8−PB2−E712D in MDCK cells, as described previously(Katsura et al., 2016), the virus titer increased during virus passagesas the proportion of Venus-positive plaques decreased (FIG. 28C).This result suggests that the loss of the Venus gene in the mutatedWT−Venus−PR8 restored the replicative efficiency of the virus.The PB2−E712D Mutation does not Cause an Appreciable Change inPolymerase Fidelity.It was hypothesized that the PB2−E712D mutation increases viralpolymerase fidelity in order to retain the inserted Venus gene duringpassages. To test this hypothesis, WT−PR8, as well as PR8−PB2−E712D,which possesses aspartic acid at position 712 of PB2 and thereforediffers from WT−PR8 by only this amino acid, were generated by reversegenetics and their mutation rates compared. Here, viruses were used thatdid not contain the Venus gene to make it easier to measure the mutationrates. PR8−PB1−V43I, which has been reported to be a high-fidelitymutant virus (Cheung et al., 2014; Naito et al., 2015), andPR8−PB1−T123A, which has been reported to be a low-fidelity mutant virus(Pauly et al., 2017), were also generated by reverse genetics and usedas controls. To estimate the mutation rates, these viruses were passagedin MDCK cells at an MOI of 0.001 and deep sequencing of the entiregenome performed; the sequencing data for the five-times passagedviruses were compared to those of viruses before passaging. The numberof nucleotide changes in the five-times passaged viruses that were notpresent before passaging were counted. The number of mutationsintroduced during the five passages are shown in FIG. 29A by segment.Also, the number of mutations per nucleotide was calculated fornormalization, and the mean values for all eight segments were compared(FIG. 29B). It was confirmed that PR8−PB1−V43I, the high-fidelitycontrol, had fewer mutations, and that PR8−PB1−T123A, the low-fidelitycontrol, had more mutations than WT−PR8. Although PR8−PB1−V43I had fewermutations than WT−PR8, the difference between PR8−PB1−V43I and WT−PR8was small. Since a previous report suggested that PB1−V43I does notalter the mutation rate (Pauly et al., 2017), the influence of PB1−V43Ion the mutation rate might be dependent on the virus strain orexperimental conditions. Moreover, there was no clear difference inmutation number between WT−PR8 and PR8−PB2−E712D. Although thepossibility that the PB2−E712D mutation affects virus polymerasefidelity cannot be excluded, the effect does not seem to be large enoughto cause an appreciable difference in Venus stability. Therefore, thisresult suggests that the stability of the Venus gene is not influencedby the fidelity of the virus polymerase.

Transcription/Replication of the Modified RNA Segment is Impaired inWT−Venus−PR8.

Some reports suggest that recombinant viruses containing a foreign geneinsertion in their NS segment can propagate more efficiently ininterferon (IFN)-deficient Vero cells than in IFN-competent cells suchas MDCK cells (Kittel et al., 2004; Ferko et al., 2001; Kuznetsova etal., 2014). Therefore, the expression levels of IFN-S in virus-infectedcells was quantified by using quantitative real-time PCR. MDCK cellswere infected with WT-Venus−PR8 or Venus−PR8−PB2−E712D at an MOI of 1 ormock infected with medium only, and the relative expression levels ofIFN-s in infected cells were quantified at 9 h postinfection.WT−Venus−PR8 induced a higher level of IFN-β expression than didVenus−PR8−PB2−E712D (FIG. 30A). This result suggests that WT-Venus−PR8does not efficiently inhibit IFN-β expression. Given that NS1 plays akey role in suppressing IFN expression and IFN-mediated antiviralresponses in the host (Garcia-Sastre et al., 1998; Opitz et al., 2007),the NS vRNA in infected cells was quantified by using influenza virusstrand-specific real-time PCR (Kawakami et al., 2011; Kupke et al.,2018). The amount of NS vRNA in WT−Venus−PR8-infected cells was 90%lower than that in Venus−PR8−PB2−E712D-infected cells (FIG. 30B),whereas there was no significant dierence in their NP vRNA expressionlevels (FIG. 30C). The NS vRNA/NP vRNA ratio in WT−Venus−PR8-infectedcells was 80% lower than that in Venus−PR8−PB2−E712D-infected cells(FIG. 30D). This result suggests that the transcription/replication ofthe NS segment is specifically impaired in WT−Venus−PR8. The sequencesof all of the plasmids used to generate viruses by reverse genetics wereconfirmed before use, and the NS segments of WT−Venus−PR8 andVenus−PR8−PB2−E712D were derived from the same NS-Venus plasmid.Therefore, it is unlikely that either WT−Venus−PR8 orVenus−PR8−PB2−E712D has a mutated promoter sequence in its NS segment.Accordingly, the difference in the transcription/replication efficiencyof the NS segment was likely caused by the PB2−E712D. Moreover, it wasconfirmed the expression level of the NS1 protein by Western blotting(FIG. 30E). Due to the reduced transcription/replication efficiency ofthe NS segment, the expression level of the NS1 protein inWT−Venus−PR8-infected cells was much lower than that inVenus−PR8−PB2−E712D-infected cells. In contrast, the expression level ofNP was almost the same. The NS1/NP ratio, quantified based on the bandintensity, was significantly reduced in WT−Venus−PR8-infected cells(FIG. 30F). It therefore appears that the low level of NS1 expressionleads to the high expression of IFN-β in WT−Venus−PR8-infected cells.Furthermore, the high expression of IFN-β causes attenuation ofWT-Venus−PR8, although it is possible that other factors are involved.

The Inserted Venus Gene is Deleted Via Internal Deletion.

To explore how deletion of the Venus gene occurs, the sequence of the NSsegment in WT−Venus−PR8 that lost Venus expression after serial passageswas determined. Plaque assays were performed using three independentlypassaged WT−Venus−PR8 virus stocks and it was found that the majority ofthe plaques were Venus negative. More than five plaques from each stockwere sequenced and it was found that one or two deletion patterns werein each virus stock. Large deletions occurred in the NS segment and mostof the Venus sequence was lost (FIG. 31A). However, no specific patternswith regard to the deletion, such as the number of nucleotide deletions,the site of the deletion(s), or specific sequences at which deletionsoccurred, were identified. It was hypothesized that the large deletionresulted from internal deletion caused by polymerase jumping, which is aknown mechanism of defective interfering viral RNA production (Davis etal., 1980; Jennings et al., 1983), or by gene recombination, which playsa role in RNA virus adaptation through rearrangement of the virus genome(Xiao et al., 2016; Simon et al., 2011; Mitanul et al., 2000;Khatchikian et al., 1989). Synonymous mutations were introduced into the3′ or 5′ region of the NS segment of WT−Venus−PR8 (FIG. 31B) and thenMDCK cells were infected at an MOI of 0.001 or 5 with each of thesemutant viruses, after which supernatants were collected at 2 days or 8 hpostinfection, respectively. The supernatant was then incubated withMDCK cells. Venus-negative plaques were picked up and amplified in MDCKcells, and then we determined the NS segment sequence in the virusesthat lost Venus expression. We found that the truncated NS segments hada synonymous mutation on only one side (FIG. 31C). No viruses were foundthat had NS segments with synonymous mutations on both sides or withouta synonymous mutation in both low- and high-MOI coinfections. Thisresult indicates that the large deletion in the NS segment resulted frominternal deletion in each NS segment and not gene recombination betweenNS segments.

Additional Mutations Stabilize the Venus Gene.

To further understand the mechanisms of the Venus deletion andstabilization, additional mutations were attempted in the polymerasecomplex that stabilizes the Venus gene. MDCK cells were infected withWT-Venus−PR8 at an MOI of 0.001, and then Venus-positive plaques werepicked and amplified in MDCK cells. After consecutive passaging andplaque purification of Venus-positive viruses, mutants were obtainedthat stably expressed enhanced Venus fluorescence. Sequence analysisrevealed that mutations were introduced into the polymerase genes PB2,PB1, and PA of each mutant (FIG. 32A). PA-180 and PA-200 are located onthe surface of the polymerase complex, as is PB2-712, whereas PB2-540,PB1-149, and PB1-684 are located inside the complex. PA-180 and PA-200are located in the endonuclease domain, PB1-149 and PB1-684 are locatednear the exit of the RNA template, and PB2-540 is located near the exitof newly synthesized RNA products (FIG. 32B), while the function of theregion around PB2-712 has remained unclear (Reich et al., 2014; Pflug etal., 2017; Gerlach et al., 2015). To determine whether these mutationscontribute to the stabilization of the Venus gene, mutant viruses weregenerated containing each of the mutations by using reverse genetics andmeasured the Venus retention ratio after four passages in MDCK cells(FIG. 32C). The mutant viruses showed enhanced Venus stability comparedto WT−Venus−PR8, indicating that these amino acids play roles in thestabilization of the Venus gene. Although further analysis is needed toclarify how these amino acids contribute to the stability of the Venusgene, considering that PB2-540, PB1-149, and PB1-684 are located nearthe polymerase internal tunnels (Reich et al., 2014; Pflug et al., 2017;Gerlach et al., 2015) that the template and product go through duringthe transcription/replication reaction, these amino acids may affect thebinding stability of the RNA template, product, and polymerase complex.Moreover, when we examined whether these mutations were found inpreviously isolated influenza A viruses in the Influenza ResearchDatabase (FIG. 32D), we found that these amino acids are extremely rare,suggesting that they are not evolutionarily beneficial.

Discussion

Recombinant influenza viruses expressing foreign genes would be usefultools; however, long insertions in virus genomes are often unstable andcause attenuation of the recombinant viruses. We previously found thatamino acids in the influenza virus polymerase complex play crucial rolesin the stabilization of foreign gene insertions; the Venus gene insertedinto the NS segment was stabilized by PB2−E712D in an H1N1 virus(Fukuyama et al., 2015; Katsura et al., 2016) and by PB2-V25A andPA-R443K in an H5N1 virus (Zhao et al., 2016). However, the mechanismsby which these amino acids contribute to the stabilization remainedunclear. In the present study, we explored the mechanism ofPB2−E712D-induced stabilization of the Venus gene inserted into the NSsegment of an H1N1 virus. It was found that thetranscription/replication efficiency of the modified segment wassignificantly reduced in WT−Venus−PR8 compared to Venus−PR8−PB2−E712D.This finding suggests that the PB2−E712D mutation stabilizes theinserted foreign gene due to the enhanced transcription/replicationefficiency of the modified RNA segment. In contrast, thetranscription/replication efficiency of segments that do not containadditional sequences is not changed in the presence or absence of thePB2−E712D mutation. Moreover, polymerase activity is reduced, notenhanced, by the PB2−E712D mutation in a minireplicon assay (Katsura etal., 2016). These results indicate that the alteration of thetranscription/replication efficiency caused by PB2−E712D is specific tomodified RNA segments. The insertion of foreign genes appears to impairthe transcription/replication of the modified segments, and thepolymerase overcomes this impairment in the presence of the PB2−E712Dmutation.

In WT−Venus−PR8, in which the Venus gene is inserted into the NSsegment, the transcription/replication efficiency of this segment issignificantly reduced. As a result, the expression of the NS1 protein isalso reduced. Since NS1 plays a role in inhibiting IFN-mediatedantiviral responses (Garcia-Sastre et al., 2008; Optiz et al., 2007)WT−Venus−PR8 cannot inhibit IFN-(3 expression efficiently, which maylead to virus attenuation. The viral titer of WT−Venus−PR8 increasesduring serial passages in MDCK cells as the virus loses Venus expression(FIGS. 28B and 28C), suggesting that mutated WT−Venus−PR8 that does notcontain the Venus gene propagates more efficiently than the originalWT−Venus−PR8. Therefore, it is likely that the immediate loss of Venusexpression in WT−Venus−PR8 results from the selection of variantswithout the Venus gene during serial passaging. Venus−PR8−PB2−E712Drestores the transcription/replication efficiency of the NS segment,leading to efficient virus replication. Therefore, viruses expressingVenus are not purged by selective pressure in the presence of thePB2−E712D mutation, which enables Venus−PR8-PB2−E712D to stably maintainthe inserted Venus gene.

How is the transcription/replication efficiency reduced on modified RNAsegments specifically, and how is it enhanced by the PB2−E712D mutation?The RNA secondary structure and the binding affinity between thepolymerase complex and the RNA templates likely hold the answer to thesequestions. Insertion of a foreign gene must change the RNA secondarystructure, and transcription/replication by the viral polymerase complexmay be negatively influenced by this unusual RNA secondary structure.Although we do not conclusively know how the PB2−E712D mutationovercomes the impairment of transcription/replication, one possibleexplanation is that the binding affinity between the polymerase complexand the RNA templates is increased.

The sequence analysis of WT−Venus−PR8 that lost Venus expression,coupled with the coinfection experiments (FIGS. 29B and 29C), suggestedthat the inserted sequence is deleted due to an internal deletion.Internal deletions often occur during influenza virus replication cyclesregardless of the presence of a foreign gene insertion and have beenreported to play roles in virus adaptation (Lui et al., 1993; Lui etal., 1985; Yang et al., 1987) and the generation of defectiveinterfering viral RNA (Davis et al., 1980; Jennings et al., 1983).Internal deletion is believed to be caused by polymerase complexdissociation from RNA templates during transcription/replication(Jennings et al., 1983; Lazarini et al., 2001; Dimmock et al., 2014;Lopez et al., 2014). Amino acid mutations in the polymerase complexaffect the frequency of occurrence of internal deletions (Fodor et al.,2003; Vasilijevic et al., 2017; Slaine et al., 2018; Te Velthuis et al.,2018). PB2−E712D may also be involved in the occurrence of internaldeletions. Therefore, the stabilization of the Venus gene inVenus−PR8−PB2−E712D may be caused not only by the enhancement of thetranscription/replication on the modified segment but also by thereduced frequency of internal deletions.

Additional mutations in the influenza virus polymerase complex wereidentified that stabilize the inserted Venus gene, which may help us tofurther understand the stabilization mechanisms based on the positionsof these mutations in the viral polymerase complex. Some of theidentified amino acids are located near the polymerase internal tunnels,which are near the RNA template or newly synthesized RNA product duringthe transcription/replication reactions (Reich et al., 2014; Pflug etal., 2017; Gerlach et al., 2015). These amino acids might directlyaffect the binding affinity between the polymerase complex, template,and product. A previous report, which showed that PB2 amino acidslocated at the template exit channel are involved in the formation ofshort aberrant RNAs (Te Velthuis et al., 2018), supports the possibilitythat amino acids near the polymerase internal tunnels affect the bindingaffinity between the polymerase complex, template, and product. However,PA-180 and PA-200, which are located at the endonuclease domain, are notnear the polymerase internal tunnels, which is also true for PB2-712.Therefore, these amino acids may affect the binding affinity indirectly,or there may be other mechanisms involved in the stabilization of theVenus gene. These mutations could be used to establish recombinantinfluenza viruses expressing a foreign gene. However, these amino acidsmay not necessarily cause the stabilization of a foreign gene in allinfluenza virus strains, since PB2-V25A, which stabilizes the Venus genein Venus−H5N1, had a negative effect on virus replication in Venus−PR8and did not cause Venus stabilization (our unpublished data).

Although the identified amino acids seem to enhance the geneticstability of virus genomes, they have been rarely found in virusisolates (FIG. 32D). It seems likely that mutations that support themaintenance of inserted sequences are not evolutionarily beneficial tothe virus. Insertions of additional sequences into virus genomes areoften deleterious for virus replication. These mutations are probablyrare in virus populations to avoid the accumulation of deleteriousinsertions. Viruses may purge deleterious insertions by reducing thetranscription/replication efficiency of RNA segments that containinsertions that form abnormal secondary structures. In conclusion,although the amino acid mutations we identified in this study are usefulfor generating recombinant viruses, they do not seem to be beneficial tothe virus in nature in the long run.

REFERENCES

-   Arilor et al., J. Virol., 86:1433 (2010).-   Avilov et al., Vaccine, 34:741 (2012).-   Basler et al., Proc. Natl. Acad. Sci. USA, 98:2746 (2001).-   Chen et al., The Lancet, 383:714 (2014).-   Dias et al., Nature, 458:914 (2009).-   Diebold et al., Science, 303:1529 (2004).-   Dos Santos Afonso et al., Virology, 341:34 (2015).-   Edgar, Nucl Acids Res., 32:1792 (2004).-   Fan et al., Virology, 384:28 (2009).-   Fujii et al., J. Virol., 79:3766 (2005).-   Fukuyama et al., Nat. Comm., 6:6600 (2015).-   Fukuyama & Kawaoka, Curr. Opin. Immunol., 23:481 (2011).-   Gabriel et al., Proc. Natl. Acad. Sci. USA, 102:18590 (2005).-   Gambotto et al., Lancet, 371:1464 (2008).-   Garcia-Sastre, Virus Res., 162:12 (2011).-   Ghaznavi et al., Annu. Rev. Pathol., 8:331 (2013).-   Go et al., BMC genomics, 13:627 (2012).-   Hatta et al., PLoS Pathog., 3:1374 (2007).-   Hatta et al., Science, 293: 1840 (2001).-   He et al., Nature, 454:1123 (2008).-   Heaton et al., J. Virol., 87:8272 (2013).-   Helft et al., J. Clin. Invest., 122:4037 (2012).-   Herold et al., J. Exp. Med., 205:3065 (2008).-   Honda & Taniguchi, Nat. Rev. Immunol., 6:644 (2006).-   Hu et al., J. Virol., 87:2660 (2013).-   Imai et al., PLoS Pathog., 6:e1001106 (2010).-   Isakova-Sivak et al., Clin. Vaccine Immunol., PMID:24648485, epub    Mar. 19 (2014).-   Itoh et al., Nature, 460:1021 (2009).-   Jiao et al., J. Virol., 82:1146 (2008).-   Jobsis, Science, 198:1264 (1977).-   Kaimal et al., Nucleic Acids Res., 38:W96 (2010).-   Kawaoka and Webster, Proc. Natl. Acad. Sci. USA, 85:324 (1988).-   Kawaoka et al., J. Virol., 63:4603 (1989).-   Kittel et al., Virology, 324:67 (2004).-   Larkin et al., Bioinform., 23:2947 (2007).-   Leung et al., Virology, 401:96 (2010).-   Li et al., J. Virol., 79:12058 (2005).-   Li et al., J. Virol., 80:11115 (2006).-   Li et al., J. Virol., 82:11880 (2008).-   Li et al., J. Virol., 84:8389 (2010).-   Li et al., N. Eng. J. Med., 370:520 (2013).-   Liu et al., Zhonghna si Yan, 26:70 (2012).-   Manicassamy et al., Proc. Natl. Acad. Sci. USA, 107:11531 (2010).-   Murakami et al., J. Virol., 82:1605 (2008).-   Naffakh et al., Annu. Rev. Microbiol., 62:403 (2008)-   Nagai et al., Nat. Biotechnol., 20:87 (2002).-   Neumann et al., Cell Res., 20:51 (2010).-   Neumann et al., Proc. Natl. Acad. Sci. USA, 96:9345 (1999).-   Obayashi et al., Nature, 454:1127 (2008).-   Ozawa et al., J. Virol., 81:30 (2007).-   Pan et al., Nature Commun., 4:2369 (2013).-   Patterson et al., J. Cell. Sci., 114:837 (2001).-   Perez et al., J. Virol., 78:3083 (2004).-   Perrone et al., PLoS Pathog., 4:e1000115 (2008).-   Pichlmair et al., Science, 314:997 (2006).-   Reed and Muench, Am. J. Hyg., 27:493 (1938).-   Regan et al., J. Virol., 80:252 (2006).-   Salomon et al., J. Exp. Med., 202:689 (2006).-   Scholtissek et al., Virology, 87:13 (1978).-   Shaner et al., Nat. Biotechnol., 22:1567 (2004).-   Shieh et al., Am. J. Pathol., 177:166 (2010).-   Shinya et al., J. Virol., 78:3083 (2004).-   Smith et al., Nature, 459:1122 (2009).-   Smyth, Stat. Appl. Genet. Mol. Biol., 3:3 (2004).-   Song et al., J. Virol., 85:2180 (2011).-   Sugiyama et al., EMBO J., 28:1803 (2009).-   Suguitan et al., J. Virol., 86:2706 (2012).-   Wang et al., PLoS One, 7:e52488 (2010).-   Watanabe et al., J. Virol., 77:10575 (2003).-   Watanabe et al., Nature, 501:551 (2013).-   Wei et al., Vaccine, 29:7163 (2011).-   Weissleder, Nature Biotech., 19:316 (2001).-   Wright and Kawaoka, Fields Virology 6th edition, (Philadelphia, Pa.,    2013).-   Yamayoshi et al., J. Virol., 88:3127 (2013).-   Yu et al., J. Virol., 85:6844 (2011).-   Zhang et al., J. Gen. Virol., 95:779 (20141-   Zhang et al., Science, 341:410 (2013).-   Zhao et al., Proteomics, 12:1970 (2012).

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification, thisinvention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details herein may be varied considerably without departing fromthe basic principles of the invention.

1. An isolated recombinant influenza virus having PA, PB1, PB2, NP, NS,M, NA, and HA viral segments, wherein i) at least one of the viralsegments is a PB2 viral segment encoding PB2 with residue at position540 that is not asparagine, a PA viral segment encoding PA with aresidue at position 180 that is not glutamine or a residue at position200 that is not threonine, or a PB1 viral segment encoding PB1 with aresidue at position 149 that is not valine, a residue at position 684that is not glutamic acid or a residue at position 685 that is notaspartic acid, or any combination thereof, wherein the recombinantinfluenza virus has enhanced stability and/or enhanced replicationrelative to a corresponding recombinant influenza virus with a residueat position 540 in PB2 that is asparagine, a residue at position 180 inPA that is glutamine, a residue at position 200 in PA that is threonine,a residue at position 149 in PB1 that is valine, a residue at position684 in PB1 that is glutamic acid or a residue at position 685 in PB1that is aspartic acid; ii) at least one of the viral segments is a PB2viral segment encoding PB2 with residue at position 540 that is notasparagine or a residue at position 712 that is not glutamic acid, andwherein at least one of the other viral segments is a PA viral segmentencoding PA with a residue at position 180 that is not glutamine or aresidue at position 200 that is not threonine, or a PB1 viral segmentencoding PB1 with a residue at position 149 that is not valine, aresidue at position 684 that is not glutamic acid or a residue atposition 685 that is not aspartic acid, or any combination thereof,wherein the recombinant influenza virus has enhanced stability and/orenhanced replication relative to a corresponding recombinant influenzavirus with a residue at position 540 in PB2 that is asparagine, aresidue in PB2 at position 712 that is glutamic acid, a residue atposition 180 in PA that is glutamine, a residue at position 200 in PAthat is threonine, a residue at position 149 in PB1 that is valine, aresidue at position 684 in PB1 that is glutamic acid or a residue atposition 685 in PB1 that is aspartic acid; or iii) the recombinant virushas two or more viral segments comprising a PB2 viral segment encodingPB2 with residue at position 540 that is not asparagine or a residue atposition 712 that is not glutamic acid, a PA viral segment encoding PAwith a residue at position 180 that is not glutamine or a residue atposition 200 that is not threonine, or a PB1 viral segment encoding PB1with a residue at position 149 that is not valine, a residue at position684 that is not glutamic acid or a residue at position 685 that is notaspartic acid, or any combination thereof, wherein the recombinantinfluenza virus has enhanced stability and/or enhanced replicationrelative to a corresponding recombinant influenza virus with a residueat position 540 in PB2 that is asparagine, a residue in PB2 at position712 that is glutamic acid, a residue at position 180 in PA that isglutamine, a residue at position 200 in PA that is threonine, a residueat position 149 in PB1 that is valine, a residue at position 684 in PB1that is glutamic acid or a residue at position 685 in PB1 that isaspartic acid.
 2. The virus of claim 1 wherein the residue at position540 of PB2 is K, R, D, E, Q, or H, the residue at position 712 of PB2 isD, N, Q, S, H, T, Y, or C, the residue at position 180 in PA is R, K, D,E, N, or H, the residue at position 200 in PA is A, I, L, C, S, M, F, P,G, or V, the residue at position 149 in PB1 is A, T, I, L, C, S, M, F,P, or G, the residue at position 684 is D, Q, S, H, T, Y, C, K, R, or N,or the residue at position 685 in PB1 is E, N, R, H, K, S, T, Y, C, orQ; the residue at position 540 of PB2 is K, R, H, D, S, H, T, Y, or C,the residue at position 712 of PB2 is D, K, H, R, Q, or N, the residueat position 180 in PA is R, K, D, N, S, H, T, Y, or H, the residue atposition 200 in PA is A, I, L, G, S, M, or V, the residue at position149 in PB1 is A, T, I, L, S, M, or G, the residue at position 684 is D,Q, H, L, R or N, or the residue at position 685 in PB1 is E, N, R, H, Kor Q; or the residue at position 540 of PB2 is K, R or H, the residue atposition 712 of PB2 is D or N, the residue at position 180 in PA is R, Kor H, the residue at position 200 in PA is A, I, L, G or V, the residueat position 149 in PB1 is A, T, I, L or G, the residue at position 684is D or N, or the residue at position 685 in PB1 is E or Q.
 3. The virusof claim 1 wherein the PB2 has a residue at position 540 that is notasparagine, the PA has a residue at position 180 that is not glutamineand a residue at position 200 that is not threonine, and the PB1 has aresidue at position 149 that is not valine, a residue at position 684that is not glutamic acid or a residue at position 685 that is notaspartic acid; the PB2 has a residue at position 540 that is notasparagine, the PA has a residue at position 180 that is not glutamineor a residue at position 200 that is not threonine, and the PB1 has aresidue at position 149 that is not valine, a residue at position 684that is not glutamic acid or a residue at position 685 that is notaspartic acid; the PB2 has a residue at position 540 that is notasparagine or a residue at 712 that is not aspartic acid, the PA has aresidue at position 180 that is not glutamine and a residue at position200 that is not threonine, and the PB1 has a residue at position 149that is not valine, a residue at position 684 that is not glutamic acidor a residue at position 685 that is not aspartic acid; or the PB2 has aresidue at position 540 that is not asparagine and a residue at 712 thatis not aspartic acid, the PA has a residue at position 180 that is notglutamine or a residue at position 200 that is not threonine, and thePB1 has a residue at position 149 that is not valine, a residue atposition 684 that is not glutamic acid or a residue at position 685 thatis not aspartic acid.
 4. The virus of claim 1 wherein the PA furthercomprises a residue at position 443 that is not arginine, the PB1further comprises a residue at position 737 that is not lysine, the PB2further comprises a residue at position 25 that is not valine or aresidue at position 712 that is not glutamic acid, the NS viral segmentencodes a NS1 with a residue at position 167 that is not proline, the HAviral segment encodes a HA with a residue at position 380 that is notthreonine, or any combination thereof.
 5. The virus of claim 4 whereinthe residue at position 443 of PA is K or H, the residue at position 737of PB1 is H or R, the residue at position 25 of PB2 is A, L, T, I, or G,the residue at position 712 of PB2 is D, the residue at position 167 ofNS1 is S, C, M, A, L, I, G or T, or any combination thereof.
 6. Thevirus of claim 1 wherein at least one of the viral segments includes aheterologous gene sequence encoding a gene product.
 7. The recombinantvirus of claim 6 wherein the heterologous sequence is in the NS viralsegment, M viral segment, NP viral segment, PA viral segment, PB1 viralsegment, or the PB2 viral segment.
 8. A vaccine having the isolatedrecombinant virus of claim
 1. 9. The vaccine of claim 8 wherein thevirus encodes a non-influenza microbial protein, a heterologousinfluenza protein or a cancer associated antigen.
 10. A plurality ofinfluenza virus vectors for preparing a reassortant, comprising a) avector for vRNA production comprising a promoter operably linked to aninfluenza virus PA DNA linked to a transcription termination sequence, avector for vRNA production comprising a promoter operably linked to aninfluenza virus PB1 DNA linked to a transcription termination sequence,a vector for vRNA production comprising a promoter operably linked to aninfluenza virus PB2 DNA linked to a transcription termination sequence,a vector for vRNA production comprising a promoter operably linked to aninfluenza virus HA DNA linked to a transcription termination sequence, avector for vRNA production comprising a promoter operably linked to aninfluenza virus NP DNA linked to a transcription termination sequence, avector for vRNA production comprising a promoter operably linked to aninfluenza virus NA DNA linked to a transcription termination sequence, avector for vRNA production comprising a promoter operably linked to aninfluenza virus M DNA linked to a transcription termination sequence,and a vector for vRNA production comprising a promoter operably linkedto an influenza virus NS cDNA linked to a transcription terminationsequence, wherein the PB1, PB2, or PA DNAs in the vectors for vRNAproduction encode at least one of: a PB2 viral segment encoding PB2 withresidue at position 540 that is not asparagine, a PA viral segmentencoding PA with a residue at position 180 that is not glutamine or aresidue at position 200 that is not threonine, or a PB1 viral segmentencoding PB1 with a residue at position 149 that is not valine, aresidue at position 684 that is not glutamic acid or a residue atposition 685 that is not aspartic acid, or a combination thereof; andoptionally b) a vector for mRNA production comprising a promoteroperably linked to a DNA segment encoding influenza virus PA, a vectorfor mRNA production comprising a promoter operably linked to a DNAsegment encoding influenza virus PB1, a vector for mRNA productioncomprising a promoter operably linked to a DNA segment encodinginfluenza virus PB2, and a vector for mRNA production comprising apromoter operably linked to a DNA segment encoding influenza virus NP,and optionally a vector for mRNA production comprising a promoteroperably linked to a DNA segment encoding influenza virus HA, a vectorfor mRNA production comprising a promoter operably linked to a DNAsegment encoding influenza virus NA, a vector for mRNA productioncomprising a promoter operably linked to a DNA segment encodinginfluenza virus M1, a vector for mRNA production comprising a promoteroperably linked to a DNA segment encoding influenza virus M2, or avector for mRNA production comprising a promoter operably linked to aDNA segment encoding influenza virus NS2.
 11. The vectors of claim 10wherein the PB1, PB2, PA, NP, NS, and M DNAs in the vectors for vRNAproduction have a sequence corresponding to one that encodes apolypeptide having at least 95% amino acid sequence identity to acorresponding polypeptide encoded by SEQ ID NOs:1-6 or 10-15.
 12. Thevectors of claim 10 wherein the residue at position 540 of PB2 is K, Ror H, the residue at position 180 in PA is R, K or H, the residue atposition 200 in PA is A, I, L, G or V, the residue at position 149 inPB1 is A, T, I, L or G, the residue at position 684 is D or N, or theresidue at position 685 in PB1 is E or Q.
 13. The vectors of claim 10wherein at least one of the viral segments includes a heterologous genesequence encoding a gene product.
 14. A method to prepare influenzavirus, comprising: contacting a cell with: a vector for vRNA productioncomprising a promoter operably linked to an influenza virus PA DNAlinked to a transcription termination sequence, a vector for vRNAproduction comprising a promoter operably linked to an influenza virusPB1 DNA linked to a transcription termination sequence, a vector forvRNA production comprising a promoter operably linked to an influenzavirus PB2 DNA linked to a transcription termination sequence, a vectorfor vRNA production comprising a promoter operably linked to aninfluenza virus HA DNA linked to a transcription termination sequence, avector for vRNA production comprising a promoter operably linked to aninfluenza virus NP DNA linked to a transcription termination sequence, avector for vRNA production comprising a promoter operably linked to aninfluenza virus NA DNA linked to a transcription termination sequence, avector for vRNA production comprising a promoter operably linked to aninfluenza virus M DNA linked to a transcription termination sequence,and a vector for vRNA production comprising a promoter operably linkedto an influenza virus NS DNA linked to a transcription terminationsequence, wherein the PB1, PB2, or PA DNAs in the vectors for vRNAproduction encode i) a PB2 with residue at position 540 that is notasparagine or a residue at position 712 that is not glutamic acid, andat least one: a PA with a residue at position 180 that is not glutamineor a residue at position 200 that is not threonine, or a PB1 with aresidue at position 149 that is not valine, a residue at position 684that is not glutamic acid or a residue at position 685 that is notaspartic acid, or any combination thereof, ii) a PB2 with residue atposition 540 that is not asparagine, a PA with a residue at position 180that is not glutamine or a residue at position 200 that is notthreonine, or a PB1 with a residue at position 149 that is not valine, aresidue at position 684 that is not glutamic acid or a residue atposition 685 that is not aspartic acid, or any combination thereof, oriii) two or more of: a PB2 with residue at position 540 that is notasparagine or a residue at position 712 that is not glutamic acid, a PAwith a residue at position 180 that is not glutamine or a residue atposition 200 that is not threonine, or a PB1 with a residue at position149 that is not valine, a residue at position 684 that is not glutamicacid or a residue at position 685 that is not aspartic acid, or anycombination thereof; and optionally a vector for mRNA productioncomprising a promoter operably linked to a DNA segment encodinginfluenza virus PA, a vector for mRNA production comprising a promoteroperably linked to a DNA segment encoding influenza virus PB1, a vectorfor mRNA production comprising a promoter operably linked to a DNAsegment encoding influenza virus PB2, and a vector for mRNA productioncomprising a promoter operably linked to a DNA segment encodinginfluenza virus NP, and optionally a vector for mRNA productioncomprising a promoter operably linked to a DNA segment encodinginfluenza virus HA, a vector for mRNA production comprising a promoteroperably linked to a DNA segment encoding influenza virus NA, a vectorfor mRNA production comprising a promoter operably linked to a DNAsegment encoding influenza virus M1, a vector for mRNA productioncomprising a promoter operably linked to a DNA segment encodinginfluenza virus M2, or a vector for mRNA production comprising apromoter operably linked to a DNA segment encoding influenza virus NS2;in an amount effective to yield infectious influenza virus.
 15. Themethod of claim 14 wherein the cell is an avian cell or a mammaliancell.
 16. The method of claim 15 wherein the cell is a Vero cell, ahuman cell or a MDCK cell.
 17. The method of claim 14 wherein thewherein the PB1, PB2, PA, NP, NS, and M DNAs in the vectors for vRNAproductions have a sequence that corresponds to one that encodes apolypeptide having at least 95% amino acid sequence identity to acorresponding polypeptide encoded by SEQ ID NOs:1-6 or 10-15.
 18. Themethod of claim 14 wherein the residue at position 540 of PB2 is K, R orH, the residue at position 712 of PB2 is D or N, the residue at position180 in PA is R, K or H, the residue at position 200 in PA is A, I, L, Gor V, the residue at position 149 in PB1 is A, T, I, L or G, the residueat position 684 is D or N, or the residue at position 685 in PB1 is E orQ.
 19. The method of claim 14 wherein the influenza virus includes aheterologous gene sequence encoding a gene product.
 20. The method ofclaim 19 wherein the heterologous sequence is 5′ or 3′ to the PA codingsequence in the PA viral segment, 5′ or 3′ to the PB1 coding sequence inthe PB1 viral segment, 5′ or 3′ to the PB2 coding sequence in the PB2viral segment or 5′ or 3′ to the NS1 coding sequence in the NS viralsegment.